Biology subjects

Till, B. J.

Publications and source records attributed to Till, B. J..

4 recordsLinked to original sources

bioRxiv2024

Phosphate transporter (Pht) gene families in rye (Secale cereale L.) - genome-wide identification and sequence diversity assessment

BackgroundPhosphorus is a macronutrient indispensable for plant growth and development. Plants utilize specialized transporters (PHT) to take up inorganic phosphorus and distribute it throughout the plant. The PHT transporters are divided into five families: PHT1 to PHT5. Each PHT family has a particular physiological and cellular function. Rye (Secale cereale L.) is a member of Triticeae, and an important source of variation for wheat breeding. It is considered to have the highest tolerance of nutrient deficiency, among Triticeae. To date, there is no report about genes involved in response to phosphorus deficiency in rye. The aim of this study was to: (i) identify and characterize putative members of different phosphate transporter families in rye, (i) assess their sequence diversity in a collection of diverse rye accessions via low-coverage resequencing (DArTreseq), and (iii) evaluate the expression of putative rye Pht genes under phosphate-deficient conditions. ResultsWe identified 29 and 35 putative Pht transporter genes in the rye Lo7 and Weining reference genomes, respectively, representing all known Pht families. Phylogenetic analysis revealed a close relationship of rye PHT with previously characterized PHT proteins from other species. Quantitative RT PCR carried out on leaf and root samples of Lo7 plants grown in Pi-deficient and control condition demonstrated that ScPht1;6, ScPht2 and ScPht3;1 are Pi-deficiency responsive. Based on DArTreseq genotyping of 94 diverse rye accessions we identified 820 polymorphic sites within rye ScPht, including 12 variants with a putatively deleterious effect. SNP density varied markedly between ScPht genes. ConclusionsThis report is the first step toward elucidating the mechanisms of ryes response to Pi deficiency. Our findings point to multiple layers of adaptation to local environments, ranging from gene copy number variation to differences in level of polymorphism across Pht family members. DArTreseq genotyping permits for a quick and cost-effective assessment of polymorphism levels across genes/gene families and supports identification and prioritization of candidates for further studies. Collectively our findings provide the foundation for selecting most promising candidates for further functional characterization.

genomics↗

bioRxiv2020

A low-cost platform suitable for sequencing-based recovery of natural variation in understudied plants.

Genetic characterization of wild and cultivated plants provides valuable knowledge for conservation and agriculture. DNA sequencing technologies are improving and costs are dropping. Yet, analysis of many species is hindered because they grow in regions that lack infrastructure for advanced molecular biology. We developed and adapted low-cost methods that address these issues. Tissue is collected and stored in silica-gel, avoiding the need for liquid nitrogen and freezers. We have optimized low-cost home-made DNA extraction to increase yields, reduce costs, and produce DNA suitable for next generation sequencing. We also describe how to build a gel documentation system for DNA quantification. As a proof of principle, we use these methods to evaluate wild Berberis darwinii, native to Southern Chile.Method summary We describe a suite of low-cost do-it-yourself methods for field collection of plant tissues, extraction of genomic DNA suitable for next generation sequencing, and home-made agarose gel documentation suitable for DNA quantification. These methods enable the collection and preparation of samples for genomic analysis in regions with limited infrastructure.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2020

Deep sampling and pooled amplicon sequencing reveals hidden genic variation in heterogeneous rye accessions

Loss of genetic variation negatively impacts breeding efforts and food security. Genebanks house over 7 million accessions representing vast allelic diversity that is a resource for sustainable breeding. Discovery of DNA variations is an important step in the efficient use of these resources. While technologies have improved and costs dropped, it remains impractical to consider resequencing millions of accessions. Candidate genes are known for most agronomic traits, providing a list of high priority targets. Heterogeneity in seed stocks means that multiple samples from an accession need to be evaluated to recover available alleles. To address this we developed a pooled amplicon sequencing approach and applied it to the out-crossing cereal rye (Secale cereale). Ninety-five rye accessions of different improvement status and worldwide origin, each represented by a pooled sample comprising DNA of 96 individual plants, were evaluated for sequence variation in six target genes involved in seed quality, biotic and abiotic stress resistance. Seventy-four predicted deleterious variants were identified using multiple algorithms. Rare variants were recovered including those found only in a low percentage of seed. A large extent of within-population heterogeneity was revealed, providing an important point for consideration during rye germplasm conservation and utilization efforts. We conclude that this approach provides a rapid and flexible method for evaluating stock heterogeneity, probing allele diversity, and recovering previously hidden variation.

genomics↗

bioRxiv2018

Fragmentation of pooled PCR products for highly multiplexed TILLING

Improvements to massively parallel sequencing have allowed the routine recovery of natural and induced sequence variants. A broad range of biological disciplines have benefited from this, ranging from plant breeding to cancer research. The need for high sequence coverage to accurately recover single nucleotide variants and small insertions and deletions limits the applicability of whole genome approaches. This is especially true in organisms with a large genome size or for applications requiring the screening of thousands of individuals, such as the reverse-genetic technique known as TILLING, where whole genome approaches become cost prohibitive. Using PCR to target and sequence chosen genomic regions provides an attractive alternative as the vast reduction in interrogated bases means that sample size can be dramatically increased through amplicon multiplexing and multidimensional sample pooling while maintaining suitable coverage for recovery of small mutations. Direct sequencing of PCR products is limited, however, due to limitations in read lengths of many next generation sequencers. In the present study we show the optimization and use of ultrasonication for the simultaneous fragmentation of multiplexed PCR amplicons for TILLING highly pooled samples. Thirty-two PCR products were produced from genomic DNA pools representing 265 pooled barley mutant lines. Mutant lines were produced with the chemical mutagen ethyl methanesulfonate. Samples were subjected to 2x300PE illumina sequencing. Evaluation of read coverage and base quality across amplicons suggests this approach is suitable for high-throughput TILLING and other applications employing highly pooled complex sampling schemes. Induced mutations previously identified in a traditional TILLING screen were recovered in this dataset further supporting the efficacy of the approach.

plant biology↗