Maha Mezghani-Khemakhem

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 subunit I sequences to assess the level and distribution of genetic diversity of A. spiraecola 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 (P = 0.70 and 0.34, respectively); however, the host‐plant had a significant effect on the partitioning of the total genetic diversity (P < 0.01). 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 subunit I (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. spiraecola 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 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.

Development of polymorphic microsatellite loci for the tomato leaf miner, Tuta absoluta (Lepidoptera: Gelechiidae)

The tomato leaf miner Tuta absoluta (Lepidoptera: Gelechiidae) is a devastating pest of tomato originating from South America (García and Espul 1982). Since its initial detection in tomato glasshouses in Spain at the end of 2006, T. absoluta has been spreading rapidly across Europe, northern Africa and Middle East, causing severe damage to tomato crops (Desneux et al. 2010; Roditakis et al. 2010; Abbes et al. 2012). The main host plant of T. absoluta is tomato (Lycopersicon esculentum). Nevertheless this pest also attacks various plants as secondary hosts such as potato (Solanum tuberosum L.), sweet pepper (S. muricatum L.), eggplant (S. melongena L.) and various wild solanaceous plants (Siqueira et al. 2000). Variable molecular markers were used to estimate genetic diversity and population structure of this invasive species. AFLP markers were used to assess genetic diversity of T. absoluta populations from Brazil (Suinaga et al. 2004). Mitochondrial and nuclear markers were also used to estimate genetic diversity of T. absoluta populations from Spain (Cifuentes et al. 2011) and, more recently, RAPD markers have been applied to understand the structure of T. absoluta populations from Tunisia to furnish information about genetic variation during the colonization process (Bettaibi et al. 2012). In recent years, microsatellites markers have proven to be very powerful in studying genetic structure of many insect populations such as Myzus persicae (MingHua et al. 2009); Aphis spiraecola (Cao et al. 2012) and Bactrocera dorsalis (Isasawin et al. 2012).

Diversity and evolution of mariner -like elements in aphid genomes

BackgroundAlthough transposons have been identified in almost all organisms, genome-wide information on mariner elements in Aphididae remains unknown. Genomes of Acyrthosiphon pisum, Diuraphis noxia and Myzus persicae belonging to the Macrosiphini tribe, actually available in databases, have been investigated.ResultsA total of 22 lineages were identified. Classification and phylogenetic analysis indicated that they were subdivided into three monophyletic groups, each of them containing at least one putative complete sequence, and several non-autonomous sublineages corresponding to Miniature Inverted-Repeat Transposable Elements (MITE), probably generated by internal deletions. A high proportion of truncated and dead copies was also detected. The three clusters can be defined from their catalytic site: (i) mariner DD34D, including three subgroups of the irritans subfamily (Macrosiphinimar, Batmar-like elements and Dnomar-like elements); (ii) rosa DD41D, found in A. pisum and D. noxia; (iii) a new clade which differs from rosa through long TIRs and thus designated LTIR-like elements. Based on its catalytic domain, this new clade is subdivided into DD40D and DD41D subgroups. Compared to other Tc1/mariner superfamily sequences, rosa DD41D and LTIR DD40-41D seem more related to maT DD37D family.ConclusionOverall, our results reveal three clades belonging to the irritans subfamily, rosa and new LTIR-like elements. Data on structure and specific distribution of these transposable elements in the Macrosiphini tribe contribute to the understanding of their evolutionary history and to that of their hosts.

A novel method for molecular targeting of insecticide resistance in Rhopalosiphum padi L. (Homoptera: Aphididae)

ABSTRACT The bird cherry-oat aphid (Rhopalosiphum padi L.) is a devastating cereal pest that develops high resistance to organophosphate and carbamate insecticides. Because acetylcholinesterase (ACE) is the target of carbamate and organophosphate insecticides, the resistance mechanism usually involves mutations occurring in ACE-encoding genes, Ace1 and Ace2. Here, we describe a novel polymerase chain reaction (PCR)-based method for the diagnosis of resistance cases associated with the point mutation F368L in the Ace2 gene. We amplified a 127 bp DNA fragment from Ace2 gene using a modified reverse primer, and digested the amplification product using SmaI endonuclease. This procedure enabled a simple and rapid distinction between resistant and susceptible genotypes for F368L mutation. Subsequently, we screened 152 R. padi samples, and found that F368L mutation occurred at low frequency, in both the homozygous (R/R) and heterozygous (R/S) states. Based upon the results of this study, we believe that molecular diagnosis of insecticide resistance should be generalized to genes and mutations involved in this process, toward an optimal accuracy of insecticide applications.

Development of New Polymorphic Microsatellite Loci for the Barley Stem Gall Midge, Mayetiola hordei (Diptera: Cecidomyiidae) from an Enriched Library

Using an enriched library method, seven polymorphic microsatellite loci were isolated from the barley stem gall midge, Mayetiola hordei. Polymorphism at loci was surveyed on 57 individual midges collected on barley in Tunisia. Across loci, polymorphism ranged from two to six alleles per locus. The observed heterozygosity varied between 0.070 and 0.877. Based on the number of alleles detected and the associated levels of heterozygosity, we believe that these loci will prove useful for population genetic studies on M. hordei.

Genome-wide identification, characterization, and evolutionary analysis of NBS-encoding resistance genes in barley

In this study, a systematic analysis of Nucleotide-Binding Site (NBS) disease resistance (R) gene family in the barley, Hordeum vulgare L. cv. Bowman, genome was performed. Using multiple computational analyses, we could identify 96 regular NBS-encoding genes and characterize them on the bases of structural diversity, conserved protein signatures, genomic distribution, gene duplications, differential expression, selection pressure, codon usage, regulation by microRNAs and phylogenetic relationships. Depending on the presence or absence of CC and LRR domains; the identified NBS genes were assigned to four distinct groups; NBS–LRR (53.1%), CC-NBS–LRR (14.6%), NBS (26%), and CC-NBS (6.3%). NBS-associated domain analysis revealed the presence of signal peptides, zinc fingers, diverse kinases, and other structural features. Eighty-five of the identified NBS-encoding genes were mapped onto the seven barley chromosomes, revealing that 50% of them were located on chromosomes 7H, 2H, and 3H, with a tendency of NBS genes to be clustered in the distal telomeric regions of the barley chromosomes. Nine gene clusters, representing 22.35% of total mapped barley NBS-encoding genes, were found, suggesting that tandem duplication stands for an important mechanism in the expansion of this gene family in barley. Phylogenetic analysis determined 31 HvNBS orthologs from rice and Brachypodium. 87 out of 96 HvNBSs were supported by expression evidence, exhibiting various and quantitatively uneven expression patterns across distinct tissues, organs, and development stages. Fourteen potential miRNA-R gene target pairs were further identified, providing insight into the regulation of NBS genes expression. These findings offer candidate target genes to engineer disease-resistant barley genotypes, and promote our understanding of the evolution of NBS-encoding genes in Poaceae crops.

In Silico Identification of MicroRNAs with B/CYDV Gene Silencing Potential

Computational investigation of a set of publicly av ailable plant microRNAs revealed 19 barleyand oth er plantsencoded miRNAs and their near-complement reverse se quences (miRNA ) that have potential to bind all B/CYDV open reading frames (ORFs) except ORF0 and ORF6. Th ese miRNAs/miRNAs , their binding positions and targets are discussed in the context of biological protecti on of cereals against B/CYDV, based on antiviral si lencing.