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Cassava is one of the most important food security crops in tropical countries, and a competitive resource for the starch, food, feed and ethanol industries. However, genomics research in this crop is much less developed compared to other economically important crops such as rice or maize. The International Center for Tropical Agriculture (CIAT) maintains the largest cassava germplasm collection in the world. Unfortunately, the genetic potential of this diversity for breeding programs remains underexploited due to the difficulties in phenotypic screening and lack of deep genomic information about the different accessions. A chromosome-level assembly of the cassava reference genome was released this year and only a handful of studies have been made, mainly to find quantitative trait loci (QTL) on breeding populations with limited variability. This work presents the results of pooled targeted resequencing of more than 1500 cassava accessions from the CIAT germplasm collection to obtain a dataset of more than 2000 variants within genes related to starch functional properties and herbicide tolerance. Results of twelve bioinformatic pipelines for variant detection in pooled samples were compared to ensure the quality of the variant calling process. Predictions of functional impact were performed using two separate methods to prioritize interesting variation for genotyping and cultivar selection. Targeted resequencing, either by pooled samples or by similar approaches such as Ecotilling or capture, emerges as a cost effective alternative to whole genome sequencing to identify interesting alleles of genes related to relevant traits within large germplasm collections. Keywords: Cassava, Pooled targeted resequencing, Herbicide tolerance, Starch biosynthesis, SNP detection

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 2×300PE 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.

Doubled haploidy is an important tool for plant breeders. It provides a rapid means of developing recombinant populations consisting of individuals that are homozygous and therefore genetically fixed. Homozygosity is also important in plant mutation breeding where many induced mutations are predicted to be recessive and mutant alleles need to be in a homozygous state before new traits are expressed. While production of doubled haploids has been described for many plant species, efficient means to validate that produced materials are indeed homozygous are needed. Polymorphism discovery methods utilizing enzymatic mismatch cleavage are ideally suited for validation of doubled haploid plants. We describe here a low-cost protocol that utilizes self-extracted single-strand-specific nucleases, standard PCR reactions and agarose gel electrophoresis that can be applied to most plant species.

Single-strand-specific nucleases are used to cleave mismatches in otherwise double-stranded DNA. Assays typically involve PCR amplification followed by a denaturing and annealing step to generate heteroduplexed DNA molecules from PCR products containing nucleotide polymorphisms. This is followed by digestion with nucleases that cleave at the site of the mismatch. The molecular weights of the cleavage products indicate the approximate position of nucleotide polymorphisms in PCR amplicons. Cleaved DNA products are observed by one of several readout platforms such as native gel electrophoresis, denaturing gel electrophoresis, capillary electrophoresis, or denaturing high-performance liquid chromatography (DHPLC). This approach is highly suitable for accurate discovery of natural and induced single-nucleotide polymorphisms (SNPs) and also small insertions and deletions (indels). The use of self-extracted single-strand-specific nucleases, typically prepared from crude extracts of celery (Apium graveolens), is common for reverse-genetics (e.g. Targeting Induced Local Lesions IN Genomes (TILLING)) and studies of natural nucleotide polymorphisms (e.g. Ecotilling). While protocols have been published describing the preparation of single-strand-specific nuclease from plant tissues, many rely on toxic chemicals, dialysis and large-volume preparatory centrifuges and also require steps to be performed at 4 °C. We provide here a streamlined room temperature extraction protocol that uses standard microcentrifuges and eliminates toxic chemicals and traditional dialysis.

Microspore embryogenesis is a method of great importance for plant production in many species. Its practical use in monocotyledonous plants, however, is limited due to the occurrence of albino plants. Molecular examinations of such albino plants showed an aberrant form of plastids and, frequently, deletions in their plastid DNA. Moreover, in our preceding work we found typical deficiencies in plastid transcription and translation. Nevertheless, there are also indications for an involvement of the nuclear genome in albino plant formation. To address these questions, we characterised, on the one hand, a plastid factor, which is possibly involved in the formation of plastid DNA deletions, and investigated, on the other hand, nuclear gene expression in albino plants. The ycf1 gene is present in the plastid genomes of dicots, while monocots only have a truncated pseudogene. The disruption of ycf1 in tobacco showed that it is essential for the survival of dicotyledonous plants. In addition, we replaced the tobacco ycf1 by the pseudogene of rice and found rearrangements in the plastid genomes of the resulting plants indicating that the ycf1 locus plays a role in the stability of plastid DNA. The study on nuclear gene expression showed that there is an extensive alteration of transcript levels in albino plants. We found that the respective genes encode proteins with a broad range of functions in plants. All these findings taken together suggest that albino plant formation is a complex phenomenon in which both, plastid and nuclear factors or their defective interactions are involved.

Background Doubled haploidy is a fundamental tool in plant breeding as it provides the fastest way to generate populations of meiotic recombinants in a genetically fixed state. A wide range of methods has been developed to produce doubled haploid (DH) plants and recent advances promise efficient DH production in otherwise recalcitrant species. Since the cellular origin of the plants produced is not always certain, rapid screening techniques are needed to validate that the produced individuals are indeed homozygous and genetically distinct from each other. Ideal methods are easily implemented across species and in crops where whole genome sequence and marker resources are limited. Results We have adapted enzymatic mismatch cleavage techniques commonly used for TILLING (Targeting Induced Local Lesions IN Genomes) for the evaluation of heterozygosity in parental, F1 and putative DH plants. We used barley as a model crop and tested 26 amplicons previously developed for TILLING. Experiments were performed using self-extracted single-strand-specific nuclease and standard native agarose gels. Eleven of the twenty-six tested primers allowed unambiguous assignment of heterozygosity in material from F1 crosses and loss of heterozygosity in the DH plants. Through parallel testing of previously developed Simple Sequence Repeat (SSR) markers, we show that 3/32 SSR markers were suitable for screening. This suggests that enzymatic mismatch cleavage approaches can be more efficient than SSR based screening, even in species with well-developed markers. Conclusions Enzymatic mismatch cleavage has been applied for mutation discovery in many plant species, including those with little or no available genomic DNA sequence information. Here, we show that the same methods provide an efficient system to screen for the production of DH material without the need of specialized equipment. This gene target based approach further allows discovery of novel nucleotide polymorphisms in candidate genes in the parental lines.