Genetic Diversity Assessment in Plants from Reduced Representation Sequencing Data.

Genetic diversity refers to the variety of genetic traits within a population or a species. It is an essential aspect of both plant ecology and plant breeding because it contributes to the adaptability, survival, and resilience of populations in changing environments. This chapter outlines a pipeline for estimating genetic diversity statistics from reduced representation or whole genome sequencing data. The pipeline involves obtaining DNA sequence reads, mapping the corresponding reads to a reference genome, calling variants from the alignments, and generating an unbiased estimation of nucleotide diversity and divergence between populations. The pipeline is suitable for single-end Illumina reads and can be adjusted for paired-end reads. The resulting pipeline provides a comprehensive approach for aligning and analyzing sequencing data to estimate genetic diversity.

Identification of Novel Induced Mutations in Seed and Vegetatively Propagated Plants from Reduced Representation or Whole Genome Sequencing Data.

Treatment of plants with chemical mutagens results primarily in the production of novel single nucleotide variants. Mutagenesis is a mostly random process and as such plants derived from mutagenesis of different seeds or in vitro material are expected to accumulate different mutations. An important step in the creation of a mutant population for forward or reverse genetics is the choice of treatment conditions (e.g., dosage) such that sufficient mutations accumulate while not adversely affecting propagation of the plant. DNA sequencing provides a quick method to evaluate the effect of different treatment conditions and their effect on the density and spectrum of accumulated mutations. Whole genome sequencing or reduced representation sequencing is carried out followed by mapping to a reference genome and production of a Variant Call Format (VCF) file. We provide here a method for generating a multi-sample VCF from mutagenized plants and describe a new tool to streamline the process of recovering unique induced mutations and determining their possible effect on gene function.

Identification of Induced Copy Number Variation from Low Coverage Sequence Data.

Induced mutations have been an important tool for plant breeding and functional genomics for more than 80 years. Novel mutations can be induced by treating seed or other plant cells with chemical mutagens or ionizing radiation. The majority of released mutant crop varieties were developed using ionizing radiation. This has been shown to create a variety of different DNA lesions including large (e.g., >=10,000 bps) copy number variations (CNV). Detection of induced DNA lesions from whole genome sequence data is useful for choosing a mutagen dosage prior to committing resources to develop a large mutant population for forward or reverse-genetic screening. Here I provide a method for detecting large induced CNV from mutant plants that utilizes a new tool to streamline the process of obtaining read coverage directly from BAM files, comparing non-mutagenized controls and mutagenized samples, and plotting the results for visual evaluation. Example data is provided from low coverage sequence data from gamma-irradiated vegetatively propagated triploid banana.

Is it the end of TILLING era in plant science?

Since its introduction in 2000, the TILLING strategy has been widely used in plant research to create novel genetic diversity. TILLING is based on chemical or physical mutagenesis followed by the rapid identification of mutations within genes of interest. TILLING mutants may be used for functional analysis of genes and being nontransgenic, they may be directly used in pre-breeding programs. Nevertheless, classical mutagenesis is a random process, giving rise to mutations all over the genome. Therefore TILLING mutants carry background mutations, some of which may affect the phenotype and should be eliminated, which is often time-consuming. Recently, new strategies of targeted genome editing, including CRISPR/Cas9-based methods, have been developed and optimized for many plant species. These methods precisely target only genes of interest and produce very few off-targets. Thus, the question arises: is it the end of TILLING era in plant studies? In this review, we recap the basics of the TILLING strategy, summarize the current status of plant TILLING research and present recent TILLING achievements. Based on these reports, we conclude that TILLING still plays an important role in plant research as a valuable tool for generating genetic variation for genomics and breeding projects.

Spectrum and Density of Gamma and X-ray Induced Mutations in a Non-Model Rice Cultivar.

Physical mutagens are a powerful tool used for genetic research and breeding for over eight decades. Yet, when compared to chemical mutagens, data sets on the effect of different mutagens and dosages on the spectrum and density of induced mutations remain lacking. To address this, we investigated the landscape of mutations induced by gamma and X-ray radiation in the most widely cultivated crop species: rice. A mutant population of a tropical upland rice, Oryza sativa L., was generated and propagated via self-fertilization for seven generations. Five dosages ranging from 75 Gy to 600 Gy in both X-ray and gamma-irradiated material were applied. In the process of a forward genetic screens, 11 unique rice mutant lines showing phenotypic variation were selected for mutation analysis via whole-genome sequencing. Thousands of candidate mutations were recovered in each mutant with single base substitutions being the most common, followed by small indels and structural variants. Higher dosages resulted in a higher accumulation of mutations in gamma-irradiated material, but not in X-ray-treated plants. The in vivo role of all annotated rice genes is yet to be directly investigated. The ability to induce a high density of single nucleotide and structural variants through mutagenesis will likely remain an important approach for functional genomics and breeding.

Generation of Mutant Plants by Gamma Ray Exposure and Development of Low-Cost TILLING Population in Chickpea (Cicer arietinum L.).

Induced mutations have been used to facilitate plant breeding for more than 80 years. Success requires the development of a mutant population and methods to evaluate that population. In this protocol we provide methods for the development of a chickpea mutant population using gamma irradiation, and low-cost methods for the molecular characterization of the mutant population. Specifically, this chapter provides detailed methods for (1) mutation induction by gamma rays and determination of LD50 and RD50, (2) phenotypic assessment of the M2 generation, (3) low-cost extraction of genomic DNA, and (4) identification of induced mutations using low-cost agarose-gel based TILLING. The methods are low-cost and designed to be applicable in most research settings.

Functional Genomics for Plant Breeding.

To face the rapidly growing world human population, an increase in agricultural productivity and production is necessary to overcome the enhanced food demand [...].

Mutagenesis and genome editing in crop improvement: perspectives for the global regulatory landscape.

Plant breeding depends on broad genetic variation. New allelic variation can be produced by targeted or random mutagenesis. Seemingly, random mutagenesis is outdated because clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology is much more precise and potentially faster. Unfortunately, genome editing is not accessible to breeders in many countries due to legal constraints. Therefore, random mutagenesis remains a vital method to create new allelic variation. Mutant offspring, however, suffer from a heavy mutation load, and application in polyploid crops is limited because multiple mutations are typically required. Exploiting random mutations became more efficient due to recent technological advancements, such as sequence-based mutant screening and genomic background selection. In this review, random and targeted mutagenesis will be compared, highlighting the legal situation.

A Bitter-Sweet Story: Unraveling the Genes Involved in Quinolizidine Alkaloid Synthesis in Lupinus albus.

Alkaloids are part of a structurally diverse group of over 21,000 cyclic nitrogen-containing secondary metabolites that are found in over 20% of plant species. Lupinus albus are naturally containing quinolizidine alkaloid (QA) legumes, with wild accessions containing up to 11% of QA in seeds. Notwithstanding their clear advantages as a natural protecting system, lupin-breeding programs have selected against QA content without proper understanding of quinolizidine alkaloid biosynthetic pathway. This review summarizes the current status in this field, with focus on the utilization of natural mutations such as the one contained in pauper locus, and more recently the development of molecular markers, which along with the advent of sequencing technology, have facilitated the identification of candidate genes located in the pauper region. New insights for future research are provided, including the utilization of differentially expressed genes located on the pauper locus, as candidates for genome editing. Identification of the main genes involved in the biosynthesis of QA will enable precision breeding of low-alkaloid, high nutrition white lupin. This is important as plant based high quality protein for food and feed is an essential for sustainable agricultural productivity.

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. The authors developed and adapted low-cost methods that address these issues. Tissue was collected and stored in silica gel, avoiding the need for liquid nitrogen and freezers. The authors optimized low-cost, homemade DNA extraction to increase yields, reduce costs and produce DNA suitable for next-generation sequencing. The authors describe how to build a gel documentation system for DNA quantification. As a proof of principle, the authors used these methods to evaluate wild Berberis darwinii, native to Southern Chile.

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

BACKGROUND: 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 L.). RESULTS: Using the amplicon sequencing approach 95 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 candidate genes with significant functions on biotic and abiotic stress resistance, and seed quality. Seventy-four predicted deleterious variants were identified using multiple algorithms. Rare variants were recovered including those found only in a low percentage of seed. CONCLUSIONS: We conclude that this approach provides a rapid and flexible method for evaluating stock heterogeneity, probing allele diversity, and recovering previously hidden variation. A large extent of within-population heterogeneity revealed in the study provides an important point for consideration during rye germplasm conservation and utilization efforts.

Genetic and comparative mapping of Lupinus luteus L. highlight syntenic regions with major orthologous genes controlling anthracnose resistance and flowering time.

Anthracnose susceptibility and ill-adapted flowering time severely affect Lupinus luteus yield, which has high seed protein content, is excellent for sustainable agriculture, but requires genetic improvement to fulfil its potential. This study aimed to (1) develop a genetic map; (2) define collinearity and regions of synteny with Lupinus angustifolius; and (3) map QTLs/candidate genes for anthracnose resistant and flowering time. A few linkage groups/genomic regions tended to be associated with segregation distortion, but did not affect the map. The developed map showed collinearity, and syntenic regions with L. angustifolius. Major QTLs were mapped in syntenic regions. Alleles from the wild parent and cultivar, explained 75% of the phenotypic variance for anthracnose resistance and 83% for early flowering, respectively. Marker sequences flanking the QTLs showed high homology with the Lanr1 gene and Flowering-locus-T of L. angustifolius. This suggests orthologous genes for both traits in the L. luteus genome. The findings are remarkable, revealing the potential to combine early flowering/anthracnose resistant in fulfilling yield capacity in L. luteus, and can be a major strategy in the genetic improvement and usage of this species for sustainable protein production. Allele sequences and PCR-marker tagging of these genes are being applied in marker assisted selection.

Comparative analyses of DNA repeats and identification of a novel Fesreba centromeric element in fescues and ryegrasses.

BACKGROUND: Cultivated grasses are an important source of food for domestic animals worldwide. Increased knowledge of their genomes can speed up the development of new cultivars with better quality and greater resistance to biotic and abiotic stresses. The most widely grown grasses are tetraploid ryegrass species (Lolium) and diploid and hexaploid fescue species (Festuca). In this work, we characterized repetitive DNA sequences and their contribution to genome size in five fescue and two ryegrass species as well as one fescue and two ryegrass cultivars. RESULTS: Partial genome sequences produced by Illumina sequencing technology were used for genome-wide comparative analyses with the RepeatExplorer pipeline. Retrotransposons were the most abundant repeat type in all seven grass species. The Athila element of the Ty3/gypsy family showed the most striking differences in copy number between fescues and ryegrasses. The sequence data enabled the assembly of the long terminal repeat (LTR) element Fesreba, which is highly enriched in centromeric and (peri)centromeric regions in all species. A combination of fluorescence in situ hybridization (FISH) with a probe specific to the Fesreba element and immunostaining with centromeric histone H3 (CENH3) antibody showed their co-localization and indicated a possible role of Fesreba in centromere function. CONCLUSIONS: Comparative repeatome analyses in a set of fescues and ryegrasses provided new insights into their genome organization and divergence, including the assembly of the LTR element Fesreba. A new LTR element Fesreba was identified and found in abundance in centromeric regions of the fescues and ryegrasses. It may play a role in the function of their centromeres.

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. 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 multi-dimensional 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. Sequencing performance was evaluated in a total of 32 pooled PCR products produced from 4096 chemically mutagenized Hordeum vulgare DNAs pooled in three dimensions. 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.

Genetic Variability Induced by Gamma Rays and Preliminary Results of Low-Cost TILLING on M2 Generation of Chickpea (Cicer arietinum L.).

In order to increase genetic variability for chickpea improvement, the Kabuli genotype, variety Ghab4, was treated with 280 Grays of gamma rays (Cobalt 60). Field characterization began with the M2 generation. A total of 135 M2 families were sown in the field resulting in approximately 4,000 plants. Traits related to phenology (days to flowering, days to maturity), plant morphology of vegetative parts (plant height, height of first pod, number of primary branches per plant) and yield (number of seeds per pod, total number of pods per plant, total number of seeds per plant, seed yield and hundred seed weight) were recorded and analyzed to evaluate genetic variability. An evaluation of the efficacy of low-cost TILLING (Targeting Induced Local Lesions IN Genomes) to discover mutations in the M2 generation was undertaken. Mutation screening focused on genes involved in resistance to two important diseases of chickpea; Ascochyta blight (AB) and Fusarium wilt (FW), as well as genes responsible for early flowering. Analysis of variance showed a highly significant difference among mutant families for all studied traits. The higher estimates of genetic parameters (genotypic and phenotypic coefficient of variation, broad sense heritability and genetic advance) were recorded for number of seeds per plant and yield. Total yield was highly significant and positively correlated with number of pods and seeds per plant. Path analysis revealed that the total number of seeds per plant had the highest positive direct effect followed by hundred seed weight parameter. One cluster from nine exhibited the highest mean values for total number of pods and seeds per plant as well as yield per plant. According to Dunnett's test, 37 M2 families superior to the control were determined for five agronomical traits. Pilot experiments with low-cost TILLING show that the seed stock used for mutagenesis is homogeneous and that small mutations do not predominate at the dosage used.

Identification of induced mutations in hexaploid wheat genome using exome capture assay.

Wheat is a staple food crop of many countries. Improving resilience to biotic and abiotic stresses remain key breeding targets. Among these, rust diseases are the most detrimental in terms of depressing wheat production. In the present study, chemical mutagenesis was used to induce mutations in the wheat variety NN-Gandum-1. This cultivar is moderately resistant to leaf and yellow rust. The aim of mutagenesis was to improve resistance to the disease as well as to study function of genes conferring resistance to the disease. In the present investigation, a 0.8% EMS dose was found optimum for supporting 45-55% germination of NN-Gandum-1. A total of 3,634 M2 fertile plants were produced from each of the M1 plant. Out of these, 33 (0.91%) and 20 plants (0.55%) showed absolute resistance to leaf and yellow rust, respectively. While 126 (3.46%) and 127 plants (3.49%) exhibited high susceptibility to the leaf and yellow rust, respectively. In the M4 generation, a total of 11 M4 lines (nine absolute resistant and two highly susceptible) and one wild type were selected for NGS-based exome capture assay. A total of 104,779 SNPs were identified that were randomly distributed throughout the wheat sub genomes (A, B and D). Induced mutations in intronic sequences predominated. The highest total number of SNPs detected in this assay were mapped to chr.2B (14,273 SNPs), which contains the highest number of targeted base pairs in the assay. The average mutation density across all regions interrogated was estimated to be one mutation per 20.91 Mb. The highest mutation frequency was found in chr.2D (1/11.7 kb) and the lowest in chr.7D (1/353.4 kb). Out of the detected mutations, 101 SNPs were filtered using analysis criteria aimed to enrich for mutations that may affect gene function. Out of these, one putative SNP detected in Lr21 were selected for further analysis. The SNP identified in chimeric allele (Lr21) of a resistant mutant (N1-252) was located in a NBS domain of chr.1BS at 3.4 Mb position. Through computational analysis, it was demonstrated that this identified SNP causes a substitution of glutamic acid with alanine, resulting in a predicted altered protein structure. This mutation, therefore, is a candidate for contributing to the resistance phenotype in the mutant line. Based on this work, we conclude that the wheat mutant resource developed is useful as a source of novel genetic variation for forward-genetic screens and also as a useful tool for gaining insights into the important biological circuits of different traits of complex genomes like wheat.

TILLING: The Next Generation.

Gene space: the final frontier in plant functional genomics. These are the voyages of TILLING, the reverse-genetics strategy that sought to boldly go where no-one had gone before by combining high-density chemical mutagenesis with high-throughput mutation discovery. Its 18-year mission has been to explore new technologies such as next generation sequencing and to seek out new strategies like in silico databases of catalogued EMS-induced mutations from entire mutant plant populations. This chapter is a clip show highlighting key milestones in the development of TILLING. Use of different technologies for the discovery of induced mutations, establishment of TILLING in different plant species, what has been learned about the effect of chemical mutagens on the plant genome, development of exome capture sequencing in wheat, and a look to the future of reverse-genetics with targeted genome editing are discussed. Graphical Abstract.

Induction and recovery of copy number variation in banana through gamma irradiation and low-coverage whole-genome sequencing.

Traditional breeding methods are hindered in bananas due to the fact that major cultivars are sterile, parthenocarpic, triploid and thus clonally propagated. This has resulted in a narrow genetic base and limited resilience to biotic and abiotic stresses. Mutagenesis of in vitro propagated bananas is one method to introduce novel alleles and broaden genetic diversity. We previously established a method for the induction and recovery of single nucleotide mutations generated with the chemical mutagen EMS. However, officially released mutant banana varieties have been created using gamma rays, a mutagen that can produce large genomic insertions and deletions (indels). Such dosage mutations may be important for generating observable phenotypes in polyploids. In this study, we establish a low-coverage whole-genome sequencing approach in triploid bananas to recover large genomic indels caused by treatment with gamma irradiation. We first evaluated the commercially released mutant cultivar 'Novaria' and found that it harbours multiple predicted deletions, ranging from 0.3 to 3.8 million base pairs (Mbp). In total, predicted deletions span 189 coding regions. To evaluate the feasibility of generating and maintaining new mutations, we developed a pipeline for mutagenesis and screening for copy number variation in Cavendish bananas using the cultivar 'Williams'. Putative mutations were recovered in 70% of lines treated with 20 Gy and 60% of the lines treated with 40 Gy. While deletion events predominate, insertions were identified in 20 Gy-treated material. Based on these results, we believe this approach can be scaled up to support large breeding projects.

Generation of Loss-of-Function Mutants for Wheat Rust Disease Resistance Gene Cloning.

One of the most important tools to identify and validate rust resistance gene function is by producing loss-of-function mutants. Mutants can be produced using irradiation, chemicals, and insertions. Among all the mutagens, ethyl methanesulfonate (EMS) and sodium azide are most favored because of the ease of use and generation of random point mutations in the genome. The mutants so produced facilitate the isolation, identification and cloning of rust resistance genes. In this chapter we describe a protocol for seed mutagenesis of wheat with EMS and sodium azide.

Next-generation sequencing (NGS)-based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population.

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next-generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty-five genes belonging to carotenoids and folate metabolism were PCR-amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600-bp amplicons were directly sequenced in a non-overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128-fold pooling. An evaluation of six different software programs (camba, crisp, gatk unified genotyper, lofreq, snver and vipr) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.