Identification of passion fruit (Passiflora edulis) chromosomes using BAC-FISH.

Passiflora edulis, the yellow passion fruit, is the main crop from the Passiflora genus, which comprises 525 species with its diversity center in South America. Genetic maps and a BAC (bacterial artificial chromosome) genomic library are available, but the nine chromosome pairs of similar size and morphology (2n = 18) hamper chromosome identification, leading to different proposed karyotypes. Thus, the aim of this study was to establish chromosome-specific markers for the yellow passion fruit using single-copy and repetitive sequences as probes in fluorescent in situ hybridizations (FISH) to allow chromosome identification and future integration with whole genome data. Thirty-six BAC clones harboring genes and three retrotransposons (Ty1-copy, Ty3-gypsy, and LINE) were selected. Twelve BACs exhibited a dispersed pattern similar to that revealed by retroelements, and one exhibited subtelomeric distribution. Twelve clones showed unique signals in terminal or subterminal regions of the chromosomes, allowing their genes to be anchored to six chromosome pairs that can be identified with single-copy markers. The markers developed herein will provide an important tool for genomic and evolutionary studies in the Passiflora genus.

Draft genome and reference transcriptomic resources for the urticating pine defoliator Thaumetopoea pityocampa (Lepidoptera: Notodontidae).

The pine processionary moth Thaumetopoea pityocampa (Lepidoptera: Notodontidae) is the main pine defoliator in the Mediterranean region. Its urticating larvae cause severe human and animal health concerns in the invaded areas. This species shows a high phenotypic variability for various traits, such as phenology, fecundity and tolerance to extreme temperatures. This study presents the construction and analysis of extensive genomic and transcriptomic resources, which are an obligate prerequisite to understand their underlying genetic architecture. Using a well-studied population from Portugal with peculiar phenological characteristics, the karyotype was first determined and a first draft genome of 537 Mb total length was assembled into 68,292 scaffolds (N50 = 164 kb). From this genome assembly, 29,415 coding genes were predicted. To circumvent some limitations for fine-scale physical mapping of genomic regions of interest, a 3X coverage BAC library was also developed. In particular, 11 BACs from this library were individually sequenced to assess the assembly quality. Additionally, de novo transcriptomic resources were generated from various developmental stages sequenced with HiSeq and MiSeq Illumina technologies. The reads were de novo assembled into 62,376 and 63,175 transcripts, respectively. Then, a robust subset of the genome-predicted coding genes, the de novo transcriptome assemblies and previously published 454/Sanger data were clustered to obtain a high-quality and comprehensive reference transcriptome consisting of 29,701 bona fide unigenes. These sequences covered 99% of the cegma and 88% of the busco highly conserved eukaryotic genes and 84% of the busco arthropod gene set. Moreover, 90% of these transcripts could be localized on the draft genome. The described information is available via a genome annotation portal (http://bipaa.genouest.org/sp/thaumetopoea_pityocampa/).

Repeat-length variation in a wheat cellulose synthase-like gene is associated with altered tiller number and stem cell wall composition.

The tiller inhibition gene (tin) that reduces tillering in wheat (Triticum aestivum) is also associated with large spikes, increased grain weight, and thick leaves and stems. In this study, comparison of near-isogenic lines (NILs) revealed changes in stem morphology, cell wall composition, and stem strength. Microscopic analysis of stem cross-sections and chemical analysis of stem tissue indicated that cell walls in tin lines were thicker and more lignified than in free-tillering NILs. Increased lignification was associated with stronger stems in tin plants. A candidate gene for tin was identified through map-based cloning and was predicted to encode a cellulose synthase-like (Csl) protein with homology to members of the CslA clade. Dinucleotide repeat-length polymorphism in the 5'UTR region of the Csl gene was associated with tiller number in diverse wheat germplasm and linked to expression differences of Csl transcripts between NILs. We propose that regulation of Csl transcript and/or protein levels affects carbon partitioning throughout the plant, which plays a key role in the tin phenotype.

Suppressed recombination and unique candidate genes in the divergent haplotype encoding Fhb1, a major Fusarium head blight resistance locus in wheat.

KEY MESSAGE: Fine mapping and sequencing revealed 28 genes in the non-recombining haplotype containing Fhb1 . Of these, only a GDSL lipase gene shows a pathogen-dependent expression pattern. Fhb1 is a prominent Fusarium head blight resistance locus of wheat, which has been successfully introgressed in adapted breeding material, where it confers a significant increase in overall resistance to the causal pathogen Fusarium graminearum and the fungal virulence factor and mycotoxin deoxynivalenol. The Fhb1 region has been resolved for the susceptible wheat reference genotype Chinese Spring, yet the causal gene itself has not been identified in resistant cultivars. Here, we report the establishment of a 1 Mb contig embracing Fhb1 in the donor line CM-82036. Sequencing revealed that the region of Fhb1 deviates from the Chinese Spring reference in DNA size and gene content, which explains the repressed recombination at the locus in the performed fine mapping. Differences in genes expression between near-isogenic lines segregating for Fhb1 challenged with F. graminearum or treated with mock were investigated in a time-course experiment by RNA sequencing. Several candidate genes were identified, including a pathogen-responsive GDSL lipase absent in susceptible lines. The sequence of the Fhb1 region, the resulting list of candidate genes, and near-diagnostic KASP markers for Fhb1 constitute a valuable resource for breeding and further studies aiming to identify the gene(s) responsible for F. graminearum and deoxynivalenol resistance.

Exploring the genome of the salt-marsh Spartina maritima (Poaceae, Chloridoideae) through BAC end sequence analysis.

Spartina species play an important ecological role on salt marshes. Spartina maritima is an Old-World species distributed along the European and North-African Atlantic coasts. This hexaploid species (2n = 6x = 60, 2C = 3,700 Mb) hybridized with different Spartina species introduced from the American coasts, which resulted in the formation of new invasive hybrids and allopolyploids. Thus, S. maritima raises evolutionary and ecological interests. However, genomic information is dramatically lacking in this genus. In an effort to develop genomic resources, we analysed 40,641 high-quality bacterial artificial chromosome-end sequences (BESs), representing 26.7 Mb of the S. maritima genome. BESs were searched for sequence homology against known databases. A fraction of 16.91% of the BESs represents known repeats including a majority of long terminal repeat (LTR) retrotransposons (13.67%). Non-LTR retrotransposons represent 0.75%, DNA transposons 0.99%, whereas small RNA, simple repeats and low-complexity sequences account for 1.38% of the analysed BESs. In addition, 4,285 simple sequence repeats were detected. Using the coding sequence database of Sorghum bicolor, 6,809 BESs found homology accounting for 17.1% of all BESs. Comparative genomics with related genera reveals that the microsynteny is better conserved with S. bicolor compared to other sequenced Poaceae, where 37.6% of the paired matching BESs are correctly orientated on the chromosomes. We did not observe large macrosyntenic rearrangements using the mapping strategy employed. However, some regions appeared to have experienced rearrangements when comparing Spartina to Sorghum and to Oryza. This work represents the first overview of S. maritima genome regarding the respective coding and repetitive components. The syntenic relationships with other grass genomes examined here help clarifying evolution in Poaceae, S. maritima being a part of the poorly-known Chloridoideae sub-family.

A glutamine-amidotransferase-like protein modulates FixT anti-kinase activity in Sinorhizobium meliloti.

BACKGROUND: Nitrogen fixation gene expression in Sinorhizobium meliloti, the alfalfa symbiont, depends on a cascade of regulation that involves both positive and negative control. On top of the cascade, the two-component regulatory system FixLJ is activated under the microoxic conditions of the nodule. In addition, activity of the FixLJ system is inhibited by a specific anti-kinase protein, FixT. The physiological significance of this negative regulation by FixT was so far unknown. RESULTS: We have isolated by random Tn5 mutagenesis a S. meliloti mutant strain that escapes repression by FixT. Complementation test and DNA analysis revealed that inactivation of an asparagine synthetase-like gene was responsible for the phenotype of the mutant. This gene, that was named asnO, encodes a protein homologous to glutamine-dependent asparagine synthetases. The asnO gene did not appear to affect asparagine biosynthesis and may instead serve a regulatory function in S. meliloti. We provide evidence that asnO is active during symbiosis. CONCLUSIONS: Isolation of the asnO mutant argues for the existence of a physiological regulation associated with fixT and makes it unlikely that fixT serves a mere homeostatic function in S. meliloti. Our data suggest that asnO might control activity of the FixT protein, in a way that remains to be elucidated. A proposed role for asnO might be to couple nitrogen fixation gene expression in S. meliloti to the nitrogen needs of the cells.

Isolation and characterization of a priB mutant of Escherichia coli influencing plasmid copy number of delta rop ColE1-type plasmids.

The lethality induced by the overproduction in Escherichia coli of a heterologous protein was used to select bacterial mutants. In one of these, the mutation responsible was mapped to priB. We describe the isolation of this mutant, the sequencing of the mutated gene, and its in vivo effect on plasmid replication.

Combined effects of the signal sequence and the major chaperone proteins on the export of human cytokines in Escherichia coli.

We have studied the export of two human proteins in the course of their production in Escherichia coli. The coding sequences of the granulocyte-macrophage colony-stimulating factor and of interleukin 13 were fused to those of two synthetic signal sequences to direct the human proteins to the bacterial periplasm. We found that the total amount of protein varies with the signal peptide-cytokine combination, as does the fraction of it that is soluble in a periplasmic extract. The possibility that the major chaperone proteins such as SecB and the GroEL-GroES and DnaK-DnaJ pairs are limiting factors for the export was tested by overexpressing one or the other of these chaperones concomitantly with the heterologous protein. The GroEL-GroES chaperone pair had no effect on protein production. Overproduction of SecB or DnaK plus DnaJ resulted in a marked increase of the quantity of human proteins in the periplasmic fraction, but this increase depends on the signal peptide-heterologous protein-chaperone association involved.