FISH landmarks reflecting meiotic recombination and structural alterations of chromosomes in wheat (Triticum aestivum L.)

Background DNA sequence composition affects meiotic recombination. However, the correlation between tandem repeat composition and meiotic recombination in common wheat (Triticum aestivum L.) is unclear. Results Non-denaturing fluorescent in situ hybridization (ND-FISH) with oligonucleotide (oligo) probes derived from tandem repeats and single-copy FISH were used to investigate recombination in three kinds of the long arm of wheat 5A chromosome (5AL). 5AL535–18/275 arm carries the tandem repeats pTa-535, Oligo-18, and pTa-275, 5AL119.2–18/275 arm carries the tandem repeats pSc119.2, Oligo-18 and pTa-275, and 5AL119.2 arm carries the tandem repeats pSc119.2. In the progeny of 5AL535–18/275 × 5AL119.2, double recombination occurred between pSc119.2 and pTa-535 clusters (119–535 interval), and between pTa-535 and Oligo-18/pTa-275 clusters (535–18 interval). The recombination rate in the 119–535 interval in the progeny of 5AL535–18/275 × 5AL119.2–18/275 was higher than that in the progeny of 5AL535–18/275 × 5AL119.2. Recombination in the 119–535 interval produced 5AL119 + 535 segments with pTa-535 and pSc119.2 tandem repeats and 5ALNo segments without these repeats. The 5AL119 + 535 and 5ALNo segments were localized between the signal sites of the single-copy probes SC5A-479 and SC5A-527. The segment between SC5A-479 and SC5A-527 in the metaphase 5ALNo was significantly longer than that in the metaphase 5AL119 + 535. Conclusion The structural variations caused by tandem repeats might be one of the factors affecting meiotic recombination in wheat. Meiotic recombination aggregated two kinds of tandemly repeated clusters into the same chromosome, making the metaphase chromosome more condensed. To conclude, our study provides a robust tool to measure meiotic recombination and select parents for wheat breeding programs.

Specific Reconstruction on pRC Plasmid to Facilitate Its Universal Chromosomal Integration in Different Ralstonia solanacearum Species Complex Strains

The pRC system is an efficient tool for genetic studies in Ralstonia solanacearum, ensuring direct insertion of foreign gene elements into Ralstonia chromosome downstream of glms. This system is designed for double recombination across glms and the downstream region, which considerably simplifies genetic studies of complementation, overexpression, pathogenicity, and in-vivo promoter activity assays with monocopy in R. solanacearum, one of the most destructive plant-pathogenic bacteria worldwide. R. solanacearum is extremely heterogeneous and is currently referred to as a Ralstonia solanacearum species complex (RSSC). The glms gene is greatly conserved, but its downstream regions are mostly different in the RSSC, which limits the application of the current pRC plasmid in the RSSC. We compared all existing 132 genome sequences in a precise genomic glms downstream region and confirmed that the pRC system is appropriate for application of chromosomal integration in all RSSC strains but needs respective reconstruction on current pRC plasmids, since glms downstream regions are greatly variable in the RSSC. RSSC strains can be grouped according to identical glms downstream regions. This grouping provides valuable information for gene insertion in this chromosomal region, as it facilitates universal application of the pRC system in RSSC strains.

A Case of Adaptive Laboratory Evolution (ALE): Biodegradation of Furfural by Pseudomonas pseudoalcaligenes CECT 5344

Pseudomonas pseudoalcaligenes CECT 5344 is a bacterium able to assimilate cyanide as a nitrogen source at alkaline pH. Genome sequencing of this strain allowed the detection of genes related to the utilization of furfurals as a carbon and energy source. Furfural and 5-(hydroxymethyl) furfural (HMF) are byproducts of sugars production during the hydrolysis of lignocellulosic biomass. Since they inhibit the yeast fermentation to obtain bioethanol from sugars, the biodegradation of these compounds has attracted certain scientific interest. P. pseudoalcaligenes was able to use furfuryl alcohol, furfural and furoic acid as carbon sources, but after a lag period of several days. Once adapted, the evolved strain (R1D) did not show any more prolonged lag phases. The transcriptomic analysis (RNA-seq) of R1D revealed a non-conservative punctual mutation (L261R) in BN5_2307, a member of the AraC family of activators, modifying the charge of the HTH region of the protein. The inactivation of the mutated gene in the evolved strain by double recombination reverted to the original phenotype. Although the bacterium did not assimilate HMF, it transformed it into value-added building blocks for the chemical industry. These results could be used to improve the production of cost-effective second-generation biofuels from agricultural wastes.

A Chromosomal Insertion Toolbox for Promoter Probing, Mutant Complementation, and Pathogenicity Studies in Ralstonia solanacearum

We describe here the construction of a delivery system for stable and directed insertion of gene constructs in a permissive chromosomal site of the bacterial wilt pathogen Ralstonia solanacearum. The system consists of a collection of suicide vectors—the Ralstonia chromosome (pRC) series—that carry an integration element flanked by transcription terminators and two sequences of homology to the chromosome of strain GMI1000, where the integration element is inserted through a double recombination event. Unique restriction enzyme sites and a GATEWAY cassette enable cloning of any promoter::gene combination in the integration element. Variants endowed with different selectable antibiotic resistance genes and promoter::gene combinations are described. We show that the system can be readily used in GMI1000 and adapted to other R. solanacearum strains using an accessory plasmid. We prove that the pRC system can be employed to complement a deletion mutation with a single copy of the native gene, and to measure transcription of selected promoters in monocopy both in vitro and in planta. Finally, the system has been used to purify and study secretion type III effectors. These novel genetic tools will be particularly useful for the construction of recombinant bacteria that maintain inserted genes or reporter fusions in competitive situations (i.e., during plant infection).