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Abstract Background De novo sequencing the entire genome of a large complex plant genome like the one of barley (Hordeum vulgare L.) is a major challenge both in terms of experimental feasibility and costs. The emergence and breathtaking progress of next generation sequencing technologies has put this goal into focus and a clone based strategy combined with the 454/Roche technology is conceivable. Results To test the feasibility, we sequenced 91 barcoded, pooled, gene containing barley BACs using the GS FLX platform and assembled the sequences under iterative change of parameters. The BAC assemblies were characterized by N50 of ~50 kb (N80 ~31 kb, N90 ~21 kb) and a Q40 of 94%. For ~80% of the clones, the best assemblies consisted of less than 10 contigs at 24-fold mean sequence coverage. Moreover we show that gene containing regions seem to assemble completely and uninterrupted thus making the approach suitable for detecting complete and positionally anchored genes. By comparing the assemblies of four clones to their complete reference sequences generated by the Sanger method, we evaluated the distribution, quality and representativeness of the 454 sequences as well as the consistency and reliability of the assemblies. Conclusion The described multiplex 454 sequencing of barcoded BACs leads to sequence consensi highly representative for the clones. Assemblies are correct for the majority of contigs. Though the resolution of complex repetitive structures requires additional experimental efforts, our approach paves the way for a clone based strategy of sequencing the barley genome.

Background: Associative transcriptomics has been used extensively in Brassica napus to enable the rapid identification of markers correlated with traits of interest. However, within the important vegetable crop species, Brassica oleracea, the use of associative transcriptomics has been limited due to a lack of fixed genetic resources and the difficulties in generating material due to self-incompatibility. Within Brassica vegetables, the harvestable product can be vegetative or floral tissues and therefore synchronisation of the floral transition is an important goal for growers and breeders. Vernalisation is known to be a key determinant of the floral transition, yet how different vernalisation treatments influence flowering in B. oleracea is not well understood., Results: Here, we present results from phenotyping a diverse set of 69 B. oleracea accessions for heading and flowering traits under different environmental conditions. We developed a new associative transcriptomics pipeline, and inferred and validated a population structure, for the phenotyped accessions. A genome-wide association study identified miR172D as a candidate for the vernalisation response. Gene expression marker association identified variation in expression of BoFLC.C2 as a further candidate for vernalisation response., Conclusions: This study describes a new pipeline for performing associative transcriptomics studies in B. oleracea. Using flowering time as an example trait, it provides insights into the genetic basis of vernalisation response in B. oleracea through associative transcriptomics and confirms its characterisation as a complex G x E trait. Candidate leads were identified in miR172D and BoFLC.C2. These results could facilitate marker-based breeding efforts to produce B. oleracea lines with more synchronous heading dates, potentially leading to improved yields., (© 2021. The Author(s).)

Background: Bread wheat has a large complex genome that makes whole genome resequencing costly. Therefore, genome complexity reduction techniques such as sequence capture make re-sequencing cost effective. With a high-quality draft wheat genome now available it is possible to design capture probe sets and to use them to accurately genotype and anchor SNPs to the genome. Furthermore, in addition to genetic variation, epigenetic variation provides a source of natural variation contributing to changes in gene expression and phenotype that can be profiled at the base pair level using sequence capture coupled with bisulphite treatment. Here, we present a new 12 Mbp wheat capture probe set, that allows both the profiling of genotype and methylation from the same DNA sample. Furthermore, we present a method, based on Agilent SureSelect Methyl-Seq, that will use a single capture assay as a starting point to allow both DNA sequencing and methyl-seq., Results: Our method uses a single capture assay that is sequentially split and used for both DNA sequencing and methyl-seq. The resultant genotype and epi-type data is highly comparable in terms of coverage and SNP/methylation site identification to that generated from separate captures for DNA sequencing and methyl-seq. Furthermore, by defining SNP frequencies in a diverse landrace from the Watkins collection we highlight the importance of having genotype data to prevent false positive methylation calls. Finally, we present the design of a new 12 Mbp wheat capture and demonstrate its successful application to re-sequence wheat., Conclusions: We present a cost-effective method for performing both DNA sequencing and methyl-seq from a single capture reaction thus reducing reagent costs, sample preparation time and DNA requirements for these complementary analyses.

Background: De novo sequencing the entire genome of a large complex plant genome like the one of barley (Hordeum vulgare L.) is a major challenge both in terms of experimental feasibility and costs. The emergence and breathtaking progress of next generation sequencing technologies has put this goal into focus and a clone based strategy combined with the 454/Roche technology is conceivable., Results: To test the feasibility, we sequenced 91 barcoded, pooled, gene containing barley BACs using the GS FLX platform and assembled the sequences under iterative change of parameters. The BAC assemblies were characterized by N50 of approximately 50 kb (N80 approximately 31 kb, N90 approximately 21 kb) and a Q40 of 94%. For approximately 80% of the clones, the best assemblies consisted of less than 10 contigs at 24-fold mean sequence coverage. Moreover we show that gene containing regions seem to assemble completely and uninterrupted thus making the approach suitable for detecting complete and positionally anchored genes.By comparing the assemblies of four clones to their complete reference sequences generated by the Sanger method, we evaluated the distribution, quality and representativeness of the 454 sequences as well as the consistency and reliability of the assemblies., Conclusion: The described multiplex 454 sequencing of barcoded BACs leads to sequence consensi highly representative for the clones. Assemblies are correct for the majority of contigs. Though the resolution of complex repetitive structures requires additional experimental efforts, our approach paves the way for a clone based strategy of sequencing the barley genome.