Evaluation of nine statistics to identify QTLs in bulk segregant analysis using next generation sequencing approaches.

BACKGROUND: Bulk segregant analysis (BSA) combined with next generation sequencing is a powerful tool to identify quantitative trait loci (QTL). The impact of the size of the study population and the percentage of extreme genotypes analysed have already been assessed. But a good comparison of statistical approaches designed to identify QTL regions using next generation sequencing (NGS) technologies for BSA is still lacking. RESULTS: We developed an R code to simulate QTLs in bulks of F2 contrasted lines. We simulated a range of recombination rates based on estimations using different crop species. The simulations were used to benchmark the ability of statistical methods identify the exact location of true QTLs. A single QTL led to a shift in allele frequency across a large fraction of the chromosome for plant species with low recombination rate. The smoothed version of all statistics performed best notably the smoothed Euclidean distance-based statistics was always found to be more accurate in identifying the location of QTLs. We propose a simulation approach to build confidence interval statistics for the detection of QTLs. CONCLUSION: We highlight the statistical methods best suited for BSA studies using NGS technologies in crops even when recombination rate is low. We also provide simulation codes to build confidence intervals and to assess the impact of recombination for application to other studies. This computational study will help select NGS-based BSA statistics that are useful to the broad scientific community.

Aquaporins are main contributors to root hydraulic conductivity in pearl millet [Pennisetum glaucum (L) R. Br.].

Pearl millet is a key cereal for food security in arid and semi-arid regions but its yield is increasingly threatened by water stress. Physiological mechanisms relating to conservation of soil water or increased water use efficiency can alleviate that stress. Aquaporins (AQP) are water channels that mediate root water transport, thereby influencing plant hydraulics, transpiration and soil water conservation. However, AQP remain largely uncharacterized in pearl millet. Here, we studied AQP function in root water transport in two pearl millet lines contrasting for water use efficiency (WUE). We observed that these lines also contrasted for root hydraulic conductivity (Lpr) and AQP contribution to Lpr. The line with lower WUE showed significantly higher AQP contribution to Lpr. To investigate AQP isoforms contributing to Lpr, we developed genomic approaches to first identify the entire AQP family in pearl millet and secondly, characterize the plasma membrane intrinsic proteins (PIP) gene expression profile. We identified and annotated 33 AQP genes in pearl millet, among which ten encoded PIP isoforms. PgPIP1-3 and PgPIP1-4 were significantly more expressed in the line showing lower WUE, higher Lpr and higher AQP contribution to Lpr. Overall, our study suggests that the PIP1 AQP family are the main regulators of Lpr in pearl millet and may possibly be associated with mechanisms associated to whole plant water use. This study paves the way for further investigations on AQP functions in pearl millet hydraulics and adaptation to environmental stresses.

An extended root phenotype: the rhizosphere, its formation and impacts on plant fitness.

Plants forage soil for water and nutrients, whose distribution is patchy and often dynamic. To improve their foraging activities, plants have evolved mechanisms to modify the physicochemical properties and microbial communities of the rhizosphere, i.e. the soil compartment under the influence of the roots. This dynamic interplay in root-soil-microbiome interactions creates emerging properties that impact plant nutrition and health. As a consequence, the rhizosphere can be considered an extended root phenotype, a manifestation of the effects of plant genes on their environment inside and/or outside of the organism. Here, we review current understanding of how plants shape the rhizosphere and the benefits it confers to plant fitness. We discuss future research challenges and how applying their solutions in crops will enable us to harvest the benefits of the extended root phenotype.

Genetic dissection of quantitative and qualitative traits using a minimum set of barley Recombinant Chromosome Substitution Lines.

BACKGROUND: Exploring the natural occurring genetic variation of the wild barley genepool has become a major target of barley crop breeding programmes aiming to increase crop productivity and sustainability in global climate change scenarios. However this diversity remains unexploited and effective approaches are required to investigate the benefits that unadapted genomes could bring to crop improved resilience. In the present study, a set of Recombinant Chromosome Substitution Lines (RCSLs) derived from an elite barley cultivar 'Harrington' as the recurrent parent, and a wild barley accession from the Fertile Crescent 'Caesarea 26-24', as the donor parent (Matus et al. Genome 46:1010-23, 2003) have been utilised in field and controlled conditions to examine the contribution of wild barley genome as a source of novel allelic variation for the cultivated barley genepool. METHODS: Twenty-eight RCSLs which were selected to represent the entire genome of the wild barley accession, were genotyped using the 9 K iSelect SNP markers (Comadran et al. Nat Genet 44:1388-92, 2012) and phenotyped for a range of morphological, developmental and agronomic traits in 2 years using a rain-out shelter with four replicates and three water treatments. Data were analysed for marker traits associations using a mixed model approach. RESULTS: We identified lines that differ significantly from the elite parent for both qualitative and quantitative traits across growing seasons and water regimes. The detailed genotypic characterisation of the lines for over 1800 polymorphic SNP markers and the design of a mixed model analysis identified chromosomal regions associated with yield related traits where the wild barley allele had a positive response increasing grain weight and size. In addition, variation for qualitative characters, such as the presence of cuticle waxes on the developing spikes, was associated with the wild barley introgressions. Despite the coarse location of the QTLs, interesting candidate genes for the major marker-trait associations were identified using the recently released barley genome assembly. CONCLUSION: This study has highlighted the role of exotic germplasm to contribute novel allelic variation by using an optimised experimental approach focused on an exotic genetic library. The results obtained constitute a step forward to the development of more tolerant and resilient varieties.

Association Mapping of Diastatic Power in UK Winter and Spring Barley by Exome Sequencing of Phenotypically Contrasting Variety Sets.

Diastatic Power (DP) is an important quality trait for malt used in adjunct brewing and distilling. Substantial genetic variation for DP exists within UK elite barley cultivars, but breeding progress has been slow due to the limited demand, compared to the overall barley market, and difficulties in assessing DP. Estimates of DP (taken from recommended and national list trials between 1994 and 2012) from a collection of UK elite winter and spring varieties were used to identify contrasting sets of high and low DP varieties. DNA samples were pooled within sets and exome capture sequencing performed. Allele frequency estimates of Single Nucleotide Polymorphisms (SNPs) identified from the sequencing were used to identify genomic locations associated with differences in DP. Individual genotypes were generated from a set of custom KASP assays, both within sets and in a wider germplasm collection, to validate allele frequency estimates and marker associations with DP. QTL identified regions previously linked to variation in DP as well as novel associations. QTL colocalised with a number of genes annotated as having a diastase related function. Results indicate that winter barley is more genetically diverse for genes influencing DP. The marker assays produced by this work represent a resource that is available for immediate use by barley breeders in the production of new high DP varieties.