Autosomal recessive congenital cataract is associated with a novel 4-bp splicing deletion mutation in a novel C10orf71 human gene.

Congenital cataract is one of the most genetically heterogeneous ocular conditions with different genes involved in its etiology. Here, we describe the analysis of a new candidate gene of a congenital bilateral cataract associated with polymalformative syndrome, moderate global developmental delay, microcephaly, axial hypotonia, intrauterine growth restriction and facial dysmorphism for two affected siblings. Molecular analysis included exome sequencing and genome wide homozygosity mapping revealed a region of homozygosity shared by the two affected siblings at 10q11.23. The new C10orf71 gene was included in this interval and direct sequencing of this gene revealed an already described homozygous c. 2123T > G mutation (p. L708R) for the two affected subjects. Interestingly, we revealed in contrast a 4-bp deletion on the 3'-splicing acceptor site of intron 3-exon 4, namely defined as IVS3-5delGCAA. The C10Orf71 gene expression analysis using RT-PCR showed an expression pattern in different fetal organs and tissues as well as in leukocytes and confirmed that the IVS3-5delGCAA deletion of the C10orf71 gene is a splicing mutation responsible for the shortening of the C10orf71 protein in the two related patients. The C10orf71 gene has not been described to date as associated to the autosomal recessive phenotype.

A PCR-based diagnostic assay for detecting DNA of the olive fruit fly, Bactrocera oleae, in the gut of soil-living arthropods.

Bactrocera oleae (Rossi) (Diptera: Tephritidae) is considered the most devastating pest of the olive tree worldwide. In an effort to develop management and biological control strategies against this pest, new molecular tools are urgently needed. In this study, we present the design of B. oleae-specific primers based on mitochondrial DNA sequences of cytochrome oxidase subunit I (COI) gene. Two pairs of B. oleae-specific primers were successfully designed and named as SBo1-F/SBo1-R and SBo2-F/SBo1-R, being able to amplify 108 and 214 bp COI fragments, respectively. The specificity of designed primers was tested by amplifying DNA from phylogenetically related (i.e. Diptera order) and other non-pest insects living in olive groves from the Mediterranean region. When using these primers on a PCR-based diagnostic assay, B. oleae DNA was detected in the gut content of a soil-living insect, Pterostichus globosus (Fabricius) (Coleoptera: Carabidae). The detection of B. oleae DNA in the guts of arthropods was further optimized by adding bovine serum albumin enhancer to the PCR reaction, in order to get a fast, reproducible and sensitive tool for detecting B. oleae remains in the guts of soil-living arthropods. This molecular tool could be useful for understanding pest-predator relationships and establishing future biological control strategies for this pest.

Members of Ensifer and Rhizobium genera are new bacterial endosymbionts nodulating Pisum sativum (L.).

A total of 84 Pisum sativum legume nodulating bacteria (LNB) were isolated from seven geographical sites from southern Tunisia. Phylogenetic analyses based on partial sequences of 16S rRNA gene and the housekeeping genes glnII, and recA grouped strains into six clusters, four of which belonged to the genus Rhizobium and two to the Ensifer genus. Among Rhizobium clusters, 41 strains were affiliated to Rhizobium leguminosarum, two strains to R. pisi, two strains to R. etli, and interestingly two strains belonged to previously undescribed Rhizobium species. The remaining two strains were closely related to Ensifer medicae (two strains) and Ensifer meliloti (two strains). A symbiotic nodC gene-based phylogeny and host specificity test showed that all Rhizobium strains nodulating pea belonged to the symbiovar viciae, whereas the Ensifer strains were associated with the symbiovar meliloti never described to date. All strains under investigation differed in the number of induced root nodules and the effectiveness of atmospheric nitrogen fixation. The R. leguminosarum PsZA23, R. leguminosarum PsGBL42, and E. medicae PsTA22a, forming the most effective symbiosis with the plant host, are potential candidates for inoculation programs.

Novel putative Mesorhizobium and Ensifer genomospecies together with a novel symbiovar psoraleae nodulate legumes of agronomic interest grown in Tunisia.

Forty rhizobial strains were isolated from Lotus creticus, L. pusillus and Bituminaria bituminosa endemic to Tunisia, and they belonged to the Mesorhizobium and Ensifer genera based on 16S rDNA sequence phylogeny. According to the concatenated recA and glnII sequence-based phylogeny, four Bituminaria isolates Pb5, Pb12, Pb8 and Pb17 formed a monophyletic group with Mesorhizobium chacoense ICMP14587T, whereas four other strains Pb1, Pb6, Pb13 and Pb15 formed two separate lineages within the Ensifer genus. Among the L. pusillus strains, Lpus9 and Lpus10 showed a 96% identical nucleotide with Ensifer meliloti CCBAU83493T; whereas six other strains could belong to previously undescribed Mesorhizobium and Ensifer species. For L. creticus strains, Lcus37, Lcus39 and Lcus44 showed 98% sequence identity with Ensifer aridi JNVU TP6, and Lcus42 shared a 96% identical nucleotide with Ensifer meliloti CCBAU83493T; whereas another four strains were divergent from all the described Ensifer and Mesorhizobium species. The analysis of the nodC gene-based phylogeny identified four symbiovar groups; Mesorhizobium sp. sv. anthyllidis (Lpus3 and Lpus11 from L. pusillus, Lcus43 from L. creticus), Ensifer medicae sv. meliloti (four strains from L. creticus and two strains from L. pusillus), E. meliloti sv. meliloti (four from L. creticus, four from L. pusillus and four from B. bituminosa). In addition, four B. bituminosa strains (Pb5, Pb8, Pb12, and Pb17) displayed a distinctive nodC sequence distant from those of other symbiovars described to date. According to their symbiotic gene sequences and host range, the B. bituminosa symbionts (Pb5, Pb8, Pb12 and Pb17) would represent a new symbiovar of M. chacoense for which sv. psoraleae is proposed.

New chromosomal lineages within Microvirga and Bradyrhizobium genera nodulate Lupinus angustifolius growing on different Tunisian soils.

Thirty-one rhizobial isolates nodulating native Lupinus angustifolius (blue lupine) plants growing in Northern Tunisian soils were isolated and analysed using different chromosomal and symbiotic gene markers. Phylogenetic analyses based on recA partial sequences grouped them into at least five groups: four of them within the genus Bradyrhizobium (26 isolates) and one into the genus Microvirga (5 isolates). Representative strains were analysed by multilocus sequence analysis of three housekeeping genes rrs-recA-glnII and rrs-gyrB-dnaK for Bradyrhizobium and Microvirga isolates, respectively. Based on this analysis, eight isolates clustered with the previously described strains Bradyrhizobium lupini USDA3051 and Bradyrhizobium canariense BTA-1. However, five of the isolates clustered separately and may constitute a new species within the Bradyrhizobium genus. The remaining five isolates were closely related to the strain Microvirga sp. LmiM8 and may constitute a new Microvirga species. The analysis of the nodC gene showed that all Bradyrhizobium strains nodulating blue lupine belong to the symbiovar genistearum, whereas the Microvirga isolates are associated with the symbiovar mediterranense. The results of this study support that the L. angustifolius root nodule symbionts isolated in Northern Tunisia belong mostly to the B. canariense/B. lupini lineages. However, new clades of Bradyrhizobium and Microvirga have been identified as L. angustifolius endosymbionts.

Assessing genotypic diversity and symbiotic efficiency of five rhizobial legume interactions under cadmium stress for soil phytoremediation.

In the framework of soil phytoremediation using local legume plants coupled with their native root-nodulating bacteria to increase forage yields and preserve contaminated soils in arid regions of Tunisia, we investigated the diversity of bacteria from root nodules of Lathyrus sativus, Lens culinaris, Medicago marina, M. truncatula, and M. minima and the symbiotic efficiency of these five legume symbiosis under Cadmium stress. Fifty bacterial strains were characterized using physiological and biochemical features such heavy metals resistant, and PCR-RFLP of 16S rDNA. Taxonomically, the isolates nodulating L. sativus, and L. culinaris are species within the genera Rhizobium and the ones associated to Medicago sp, within the genera Sinorhizobium. The results revealed also that the cadmium tolerance of the different legumes-rhizobia interaction was as follows: M. minima < M. truncatula < M. marina < L. sativus < L. culinaris indicating that the effect of Cadmium on root nodulation and biomass production is more deleterious on M. minima-S. meliloti and M. truncatula-S. meliloti than in other symbiosis. Knowledge on genetic and functional diversity of M. marina, L. sativus and L. culinaris microsymbiotes is very useful for inoculant strain selection and can be selected to develop inoculants for soil phytoremediation.