A systems genetics approach to deciphering the effect of dosage variation on leaf morphology in Populus.

Gene copy number variation is frequent in plant genomes of various species, but the impact of such gene dosage variation on morphological traits is poorly understood. We used a large population of Populus carrying genomically characterized insertions and deletions across the genome to systematically assay the effect of gene dosage variation on a suite of leaf morphology traits. A systems genetics approach was used to integrate insertion and deletion locations, leaf morphology phenotypes, gene expression, and transcriptional network data, to provide an overview of how gene dosage influences morphology. Dosage-sensitive genomic regions were identified that influenced individual or pleiotropic morphological traits. We also identified cis-expression quantitative trait loci (QTL) within these dosage QTL regions, a subset of which modulated trans-expression QTL as well. Integration of data types within a gene co-expression framework identified co-expressed gene modules that are dosage sensitive, enriched for dosage expression QTL, and associated with morphological traits. Functional description of these modules linked dosage-sensitive morphological variation to specific cellular processes, as well as candidate regulatory genes. Together, these results show that gene dosage variation can influence morphological variation through complex changes in gene expression, and suggest that frequently occurring gene dosage variation has the potential to likewise influence quantitative traits in nature.

A comprehensive genomic scan reveals gene dosage balance impacts on quantitative traits in Populus trees.

Gene dosage variation and the associated changes in gene expression influence a wide variety of traits, ranging from cancer in humans to yield in plants. It is also expected to affect important traits of ecological and agronomic importance in forest trees, but this variation has not been systematically characterized or exploited. Here we performed a comprehensive scan of the Populus genome for dosage-sensitive loci affecting quantitative trait variation for spring and fall phenology and biomass production. The study population was a large collection of clonally propagated F1 hybrid lines of Populus that saturate the genome 10-fold with deletions and insertions (indels) of known sizes and positions. As a group, the phenotypic means of the indel lines consistently differed from control nonindel lines, with an overall negative effect of both insertions and deletions on all biomass-related traits but more diverse effects and an overall wider phenotypic distribution of the indel lines for the phenology-related traits. We also investigated the correlation between gene dosage at specific chromosomal locations and phenotype, to identify dosage quantitative trait loci (dQTL). Such dQTL were detected for most phenotypes examined, but stronger effect dQTL were identified for the phenology-related traits than for the biomass traits. Our genome-wide screen for dosage sensitivity in a higher eukaryote demonstrates the importance of global genomic balance and the impact of dosage on life history traits.

Efficient Genome-Wide Detection and Cataloging of EMS-Induced Mutations Using Exome Capture and Next-Generation Sequencing.

Chemical mutagenesis efficiently generates phenotypic variation in otherwise homogeneous genetic backgrounds, enabling functional analysis of genes. Advances in mutation detection have brought the utility of induced mutant populations on par with those produced by insertional mutagenesis, but systematic cataloguing of mutations would further increase their utility. We examined the suitability of multiplexed global exome capture and sequencing coupled with custom-developed bioinformatics tools to identify mutations in well-characterized mutant populations of rice (Oryza sativa) and wheat (Triticum aestivum). In rice, we identified ∼18,000 induced mutations from 72 independent M2 individuals. Functional evaluation indicated the recovery of potentially deleterious mutations for >2600 genes. We further observed that specific sequence and cytosine methylation patterns surrounding the targeted guanine residues strongly affect their probability to be alkylated by ethyl methanesulfonate. Application of these methods to six independent M2 lines of tetraploid wheat demonstrated that our bioinformatics pipeline is applicable to polyploids. In conclusion, we provide a method for developing large-scale induced mutation resources with relatively small investments that is applicable to resource-poor organisms. Furthermore, our results demonstrate that large libraries of sequenced mutations can be readily generated, providing enhanced opportunities to study gene function and assess the effect of sequence and chromatin context on mutations.

De novo transcriptome sequencing in Bixa orellana to identify genes involved in methylerythritol phosphate, carotenoid and bixin biosynthesis.

BACKGROUND: Bixin or annatto is a commercially important natural orange-red pigment derived from lycopene that is produced and stored in seeds of Bixa orellana L. An enzymatic pathway for bixin biosynthesis was inferred from homology of putative proteins encoded by differentially expressed seed cDNAs. Some activities were later validated in a heterologous system. Nevertheless, much of the pathway remains to be clarified. For example, it is essential to identify the methylerythritol phosphate (MEP) and carotenoid pathways genes. RESULTS: In order to investigate the MEP, carotenoid, and bixin pathways genes, total RNA from young leaves and two different developmental stages of seeds from B. orellana were used for the construction of indexed mRNA libraries, sequenced on the Illumina HiSeq 2500 platform and assembled de novo using Velvet, CLC Genomics Workbench and CAP3 software. A total of 52,549 contigs were obtained with average length of 1,924 bp. Two phylogenetic analyses of inferred proteins, in one case encoded by thirteen general, single-copy cDNAs, in the other from carotenoid and MEP cDNAs, indicated that B. orellana is closely related to sister Malvales species cacao and cotton. Using homology, we identified 7 and 14 core gene products from the MEP and carotenoid pathways, respectively. Surprisingly, previously defined bixin pathway cDNAs were not present in our transcriptome. Here we propose a new set of gene products involved in bixin pathway. CONCLUSION: The identification and qRT-PCR quantification of cDNAs involved in annatto production suggest a hypothetical model for bixin biosynthesis that involve coordinated activation of some MEP, carotenoid and bixin pathway genes. These findings provide a better understanding of the mechanisms regulating these pathways and will facilitate the genetic improvement of B. orellana.

A systems genetic analysis identifies putative mechanisms and candidate genes regulating vessel traits in poplar wood.

Wood is the water conducting tissue of tree stems. Like most angiosperm trees, poplar wood contains water-conducting vessel elements whose functional properties affect water transport and growth rates, as well as susceptibility to embolism and hydraulic failure during water stress and drought. Here we used a unique hybrid poplar pedigree carrying genomically characterized chromosomal insertions and deletions to undertake a systems genomics analysis of vessel traits. We assayed gene expression in wood forming tissues from clonal replicates of genotypes covering dosage quantitative trait loci with insertions and deletions, genotypes with extreme vessel trait phenotypes, and control genotypes. A gene co-expression analysis was used to assign genes to modules, which were then used in integrative analyses to identify modules associated with traits, to identify putative molecular and cellular processes associated with each module, and finally to identify candidate genes using multiple criteria including dosage responsiveness. These analyses identified known processes associated with vessel traits including stress response, abscisic acid and cell wall biosynthesis, and in addition identified previously unexplored processes including cell cycle and protein ubiquitination. We discuss our findings relative to component processes contributing to vessel trait variation including signaling, cell cycle, cell expansion, and cell differentiation.

A k-mer-based bulked segregant analysis approach to map seed traits in unphased heterozygous potato genomes.

High-throughput sequencing-based methods for bulked segregant analysis (BSA) allow for the rapid identification of genetic markers associated with traits of interest. BSA studies have successfully identified qualitative (binary) and quantitative trait loci (QTLs) using QTL mapping. However, most require population structures that fit the models available and a reference genome. Instead, high-throughput short-read sequencing can be combined with BSA of k-mers (BSA-k-mer) to map traits that appear refractory to standard approaches. This method can be applied to any organism and is particularly useful for species with genomes diverged from the closest sequenced genome. It is also instrumental when dealing with highly heterozygous and potentially polyploid genomes without phased haplotype assemblies and for which a single haplotype can control a trait. Finally, it is flexible in terms of population structure. Here, we apply the BSA-k-mer method for the rapid identification of candidate regions related to seed spot and seed size in diploid potato. Using a mixture of F1 and F2 individuals from a cross between 2 highly heterozygous parents, candidate sequences were identified for each trait using the BSA-k-mer approach. Using parental reads, we were able to determine the parental origin of the loci. Finally, we mapped the identified k-mers to a closely related potato genome to validate the method and determine the genomic loci underlying these sequences. The location identified for the seed spot matches with previously identified loci associated with pigmentation in potato. The loci associated with seed size are novel. Both loci are relevant in future breeding toward true seeds in potato.

Discovery of rare mutations in populations: TILLING by sequencing.

Discovery of rare mutations in populations requires methods, such as TILLING (for Targeting Induced Local Lesions in Genomes), for processing and analyzing many individuals in parallel. Previous TILLING protocols employed enzymatic or physical discrimination of heteroduplexed from homoduplexed target DNA. Using mutant populations of rice (Oryza sativa) and wheat (Triticum durum), we developed a method based on Illumina sequencing of target genes amplified from multidimensionally pooled templates representing 768 individuals per experiment. Parallel processing of sequencing libraries was aided by unique tracer sequences and barcodes allowing flexibility in the number and pooling arrangement of targeted genes, species, and pooling scheme. Sequencing reads were processed and aligned to the reference to identify possible single-nucleotide changes, which were then evaluated for frequency, sequencing quality, intersection pattern in pools, and statistical relevance to produce a Bayesian score with an associated confidence threshold. Discovery was robust both in rice and wheat using either bidimensional or tridimensional pooling schemes. The method compared favorably with other molecular and computational approaches, providing high sensitivity and specificity.

Efficient construction of a linkage map and haplotypes for Mentha suaveolens using sequence capture.

The sustainability of many crops is hindered by the lack of genomic resources and a poor understanding of natural genetic diversity. Particularly, application of modern breeding requires high-density linkage maps that are integrated into a highly contiguous reference genome. Here, we present a rapid method for deriving haplotypes and developing linkage maps, and its application to Mentha suaveolens, one of the diploid progenitors of cultivated mints. Using sequence-capture via DNA hybridization to target single nucleotide polymorphisms (SNPs), we successfully genotyped ∼5000 SNPs within the genome of >400 individuals derived from a self cross. After stringent quality control, and identification of nonredundant SNPs, 1919 informative SNPs were retained for linkage map construction. The resulting linkage map defined a total genetic space of 942.17 cM divided among 12 linkage groups, ranging from 56.32 to 122.61 cM in length. The linkage map is in good agreement with pseudomolecules from our preliminary genome assembly, proving this resource effective for the correction and validation of the reference genome. We discuss the advantages of this method for the rapid creation of linkage maps.

Diploid mint (M. longifolia) can produce spearmint type oil with a high yield potential.

Mint oil is a key source of natural flavors with wide industrial applications. Two unbalanced polyploid cultivars named Native (Mentha Spicata L) and Scotch (M. × gracilis Sole) are the main producers of spearmint type oil, which is characterized by high levels of the monoterpenes (-)-carvone and (-)-limonene. These cultivars have been the backbone of spearmint oil production for decades, while breeding and improvement remained largely unexplored, in part, due to sterility in cultivated lines. Here we show that sexual breeding at the diploid level can be leveraged to develop new varieties that produce spearmint type oil, along with the improvement of other important traits. Using field trials and GC-FID oil analysis we characterized plant materials from a public germplasm repository and identified a diploid accession that exhibited 89.5% increase in oil yield, compared to the industry standard, and another that produces spearmint type oil. Spearmint-type oil was present at high frequency in a segregating F2 population (32/160) produced from these two accessions. Field-testing of ten of these F2 lines showed segregation for oil yield and confirmed the production of spearmint-type oil profiles. Two of these lines combined high yield and spearmint-type oil with acceptable analytic and sensory profiles. These results demonstrate that spearmint-type oil can be produced in a diploid background with high yield potential, providing a simpler genetic system for the development of improved spearmint varieties.

Creation and Genomic Analysis of Irradiation Hybrids in Populus.

Establishing efficient functional genomic systems for creating and characterizing genetic variation in forest trees is challenging. Here we describe protocols for creating novel gene-dosage variation in Populus through gamma-irradiation of pollen, followed by genomic analysis to identify chromosomal regions that have been deleted or inserted in each F1 individual. Irradiated pollen is used in a controlled, interspecific cross to create F1 progeny that carry deletions and insertions of chromosomal regions. These insertions and deletions result in novel changes in gene dosage that in turn affect both qualitative and quantitative phenotypic traits. The protocols described here outline the processes involved in optimizing irradiation levels and performing controlled crosses, sowing seed and propagating seedlings, and genome-wide sequence-based analysis of deletions and insertions in the F1 progeny. The same approach could be applied to other vegetatively propagated species. © 2016 by John Wiley & Sons, Inc.

Tilling by sequencing.

TILLING is a method to find mutations in a gene of interest by scanning amplicons from a mutagenized population for sequence changes, commonly a single nucleotide. In the past 5 years, mutation detection by sequencing has become increasingly popular. This chapter details the experimental flow for TILLING-by-Sequencing, highlighting the critical steps involved in tridimensional pooling of genomic DNA templates, preparation of libraries for high-throughput sequencing, and bioinformatic processing of the sequence data.

Perturbation of parentally biased gene expression during interspecific hybridization.

Interspecific hybridization often induces epigenetic remodeling that leads to transposon activation, gene expression changes, and loss of imprinting. These genomic changes can be deleterious and contribute to postzygotic hybrid incompatibility. In Arabidopsis, loss of genomic imprinting of PHERES1 and presumed failure of Polycomb Repressive Complex contributes to seed inviability observed in A. thaliana X A. arenosa interspecific hybrids. We used this species pair to further analyze the relationship between parentally biased gene expression and postzygotic hybrid incompatibility using two A. thaliana accessions, Col-0 and C24, with differential seed survival. We found that parentally biased expression was perturbed to a similar degree in both A. thaliana hybrids for PHERES1, HDG3, and six other normally paternally expressed genes. We propose that early genome remodeling and loss of imprinting of seed development genes induces lethality in both compatible and incompatible hybrids.

High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture.

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA-genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.