Telomere-mediated truncation of barley chromosomes

Abstract

Engineered minichromosomes offer an enormous opportunity to plant biotechnology as they have the potential to simultaneously transfer and stably express multiple genes. Following a top-down approach, we truncated endogenous chromosomes in barley (Hordeum vulgare) by Agrobacterium-mediated transfer of T-DNA constructs containing telomere sequences. Blocks of Arabidopsis-like telomeric repeats were inserted into a binary vector suitable for stable transformation. After transfer of these constructs into immature embryos of diploid and tetraploid barley, chromosome truncation by T-DNA-induced de novo formation of telomeres could be confirmed by fluorescent in situ hybridisation, primer extension telomere repeat amplification and DNA gel blot analysis in regenerated plants. Telomere seeding connected to chromosome truncation was found in tetraploid plants only, indicating that genetic redundancy facilitates recovery of shortened chromosomes. Truncated chromosomes were transmissible in sexual reproduction, but were inherited at rates lower than expected according to Mendelian rules.

Engineered plant minichromosomes

“…In maize, minichromosomes generated by transgene-mediated telomere seeding in A chromosomes were transmitted through meiosis to 33% of the progeny obtained by self-pollination or to 12% to 39% of progeny via male gametes (Yu et al, 2007), a rate similar to minichromosomes generated by breakage-fusion-bridge cycles (Kato et al, 2005). Transmission of truncated chromosomes was also below the rates expected from the Mendelian rules in progenies obtained by self-pollination in other plant species, accounting for 52% to 72% for tetraploid A. thaliana (Teo et al, 2011) and 54% for tetraploid barley (Kapusi et al, 2012). Therefore, to ensure stable heritability, it seems important that genes are identified that code for genetic features linked with a high meiotic transmissibility of engineered chromosomes, such as by increased pairing and/or crossover frequency, and are included together with the genes for desired traits.…”

Minichromosomes: Vectors for Crop Improvement

“…In maize, a truncated A chromosome was rescued in a spontaneous tetraploidy event [39]. Tetraploid plants have also been used for A chromosome truncation in Arabidopsis thaliana [40] and barley (Hordeum vulgare) [41]. The truncated chromosome can then be transferred to a diploid background by successive crosses of the tetrapliod by a diploid to produce a triploid and again by a diploid to recover the truncated minichromosome as an extra entity in a diploid background [39].…”

Recent Advancements in Gene Expression and Enabling Technologies in Crop Plants

“…This technique is effective on both normal A and supernumerary B chromosomes. Telomere-mediated truncation in plants was first demonstrated in maize but has also been shown to occur in Arabidopsis (Nelson et al 2011;Teo et al 2011), barley (Kapusi et al 2012), and rice .…”

“…First, tetraploids can be used as the target of truncation. This was first found to occur in maize ) and then was used intentionally in Arabidopsis (Nelson et al 2011;Teo et al 2011) and barley (Kapusi et al 2012). In this case, a truncated chromosome can be recovered because in a tetraploid plant, other copies of the homologous chromosome will be present in the gametophytes and will supply the gene functions otherwise missing in the truncated chromosome.…”