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M ycobacterium tuberculosis isolates of the Beijing 94-32 cluster (also named the Central Asian/Russian Beijing strain) constitute an important component of the population structure of the pathogen in the countries of the Former Soviet Union (1-4). A variable-number tandem-repeat (VNTR)-based analysis suggested that this genotype could speculatively trace its origins to the northwestern regions of China (5). Beijing 94-32 is the largest type within the VNTR-defined CC1 group (6) and falls within the East Europe 1 group as defined by whole-genome sequencing (WGS) (7). Our analysis of all CC1 isolates compiled in the work of Merker et al. (6) demonstrated that type 94-32 presents the largest node in the central position in the phylogenetic network (see Fig. S1 in the supplemental material), and we therefore suggest naming this clonal complex the Beijing 94-32 cluster. The 94-32 cluster isolates were associated with multidrug-resistant/extremely drug-resistant tuberculosis in Russia (8) and in Uzbekistan (termed the Central Asia outbreak strain [2]), and in immigrants in Western Europe (9, 10). This justifies the interest in having a simple tool to rapidly detect this clinically and epidemiologically relevant strain. In this study, DNA of 19 Russian M. tuberculosis isolates of the Beijing genotype was subjected to WGS on the MiSeq platform (Illumina). The next-generation sequencing (NGS) data were deposited in the NCBI Sequence Read Archive (project number PRJNA305488). The fastq and vcf files were subjected to comprehensive bioinformatics

BackgroundThe relatively low efficiency of biolistic transformation and subsequent integration of multiple copies of the introduced gene/s significantly complicate the genetic modification of wheat (Triticum aestivum) and other plant species. One of the key factors contributing to the reproducibility of this method is the uniformity of the DNA/gold suspension, which is dependent on the coating procedure employed. It was also shown recently that the relative frequency of single copy transgene inserts could be increased through the use of nanogram quantities of the DNA during coating.ResultsA simplified DNA/gold coating method was developed to produce fertile transgenic plants, via microprojectile bombardment of callus cultures induced from immature embryos. In this method, polyethyleneglycol (PEG) and magnesium salt solutions were utilized in place of the spermidine and calcium chloride of the standard coating method, to precipitate the DNA onto gold microparticles. The prepared microparticles were used to generate transgenics from callus cultures of commercial bread wheat cv. Gladius resulting in an average transformation frequency of 9.9%. To increase the occurrence of low transgene copy number events, nanogram amounts of the minimal expression cassettes containing the gene of interest and the hpt gene were used for co-transformation. A total of 1538 transgenic wheat events were generated from 15,496 embryos across 19 independent experiments. The variation of single copy insert frequencies ranged from 16.1 to 73.5% in the transgenic wheat plants, which compares favourably to published results.ConclusionsThe DNA/gold coating procedure presented here allows efficient, large scale transformation of wheat. The use of nanogram amounts of vector DNA improves the frequency of single copy transgene inserts in transgenic wheat plants.