Joanna Jankowicz-Cieslak

Induction, rapid fixation and retention of mutations in vegetatively propagated banana

Mutation discovery technologies have enabled the development of reverse genetics for many plant species and allowed sophisticated evaluation of the consequences of mutagenesis. Such methods are relatively straightforward for seed-propagated plants. To develop a platform suitable for vegetatively propagated species, we treated isolated banana shoot apical meristems with the chemical mutagen ethyl methanesulphonate, recovered plantlets and screened for induced mutations. A high density of GC-AT transition mutations were recovered, similar to that reported in seed-propagated polyploids. Through analysis of the inheritance of mutations, we observed that genotypically heterogeneous stem cells resulting from mutagenic treatment are rapidly sorted to fix a single genotype in the meristem. Further, mutant genotypes are stably inherited in subsequent generations. Evaluation of natural nucleotide variation showed the accumulation of potentially deleterious heterozygous alleles, suggesting that mutation induction may uncover recessive traits. This work therefore provides genotypic insights into the fate of totipotent cells after mutagenesis and suggests rapid approaches for mutation-based functional genomics and improvement of vegetatively propagated crops.

Geographic origin is not supported by the genetic variability found in a large living collection of Jatropha curcas with accessions from three continents

Increasing economic interest in Jatropha curcas requires a major research focus on the genetic background and geographic origin of this non-edible biofuel crop. To determine the worldwide genetic structure of this species, amplified fragment length polymorphisms, inter simple sequence repeats, and novel single nucleotide polymorphisms (SNPs) were employed for a large collection of 907 J. curcas accessions and related species (RS) from three continents, 15 countries and 53 regions. PCoA, phenogram, and cophenetic analyses separated RS from two J. curcas groups. Accessions from Mexico, Bolivia, Paraguay, Kenya, and Ethiopia with unknown origins were found in both groups. In general, there was a considerable overlap between individuals from different regions and countries. The Bayesian approach using structure demonstrated two groups with a low genetic variation. Analysis of molecular varience revealed significant variation among individuals within populations. SNPs found by in silico analyses of Δ12 fatty acid desaturase indicated possible changes in gene expression and thus in fatty acid profiles. SNP variation was higher in the curcin gene compared to genes involved in oil production. Novel SNPs allowed separating toxic, non-toxic, and Mexican accessions. The present study confirms that human activities had a major influence on the genetic diversity of J. curcas, not only because of domestication, but also because of biased selection.

Forward and Reverse Genetics in Crop Breeding

The ability to identify, study, create and utilize genetic variation are arguably fundamental aspects in the development of human beings. Traditional, or forward, genetics has its roots in man’s first attempts at the domestication of plants and animals. Novel and preferred traits such as the lack of seed shattering were selected from large populations and maintained for future propagation, leading to the first cultivars. The discovery of DNA as the heritable material thousands of years later enabled the development of reverse genetics whereby targeted lesions in the genome are recovered to test and utilize functional variation in genes. A major contributor to both forward and reverse genetics was the discovery in the early twentieth century that mutations can be induced in genomes at frequencies in several orders of magnitude higher than typically observable in nature. The ability to produce novel variation has fueled the development of thousands of new crop cultivars. Examples exist of increased disease resistance, higher yields, tolerance to abiotic stresses such as drought and salinity and improved nutritional quality. In an era where global food security is threatened by climate change and variation and growing populations, use of induced mutations is an important method in the breeder’s toolbox. This review describes the use of induced mutations for forward and reverse genetics in plants, with a focus on crops. Different mutagens and random versus targeted approaches are described. Additionally, newly emerging methods and technologies are discussed that promise to advance basic and applied plant sciences.

Validation of doubled haploid plants by enzymatic mismatch cleavage

BackgroundDoubled 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.ResultsWe 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.ConclusionsEnzymatic 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.

Mutation and mutation screening.

Molecular techniques have created the opportunity for great advances in plant mutation genetics and the science of mutation breeding. The powerful targeted induced local lesions in genomes (TILLING) technique has introduced the possibility of reverse genetics-the ability to screen for mutations at the DNA level prior to assessing phenotype. Fundamental to TILLING is the induction of mutant populations (or alternatively, the identification of mutants in the environment); and mutation induction requires an understanding and assessment of the appropriate mutagen dose required. The techniques of mutation induction, dose optimization, and TILLING are explained.

The Use of EcoTILLING for the Genetic Improvement of Jatropha curcas L.

Distinguishing adaptive variation from neutral mutations are becoming key issues to understand heritable phenotypic changes and to carry out functional genomics. Genetic diversity, most commonly manifested as Single Nucleotide Polymorphisms (SNPs), can provide clues to the adaptive processes and the population histories that have played a role in the evolution of tree species, such as Jatropha. To efficiently identify SNPs, a number of different techniques have been developed, which all have their limitations. Thus, there is an increasing demand for high-throughput genotyping technologies to increase our ability to determine rare nucleotide differences for traits of economical interest. Reverse genetics approaches rely on the detection of differences in target sequences to identify allelic variations in natural or mutant populations and in the context of functional genomics. EcoTILLING, a variation of Targeting Induced Local Lesions IN Genomes (TILLING), allows high-throughput analyses of natural genetic diversity in plants, particularly in species with limited genetic diversity such as Jatropha curcas. To achieve certain breeding goals in J. curcas, the use of germplasm from different regions is necessary. Understanding the population structure of J. curcas is challenging due to limited genetic variability and information on phylogenetic relationships between accessions of J. curcas and related species. In fact, the genetic map of J. curcas is not well-developed and research on molecular markers is just expanding that could be used to clearly distinguish world wide accessions. Therefore, a resource database of SNPs in J. curcas will give researchers and breeders a tool for answering questions related to population structure, adaptation, and gene function. The development of cultivars of J. curcas by conventional breeding will profit largely from biotechnological support (pathogen-free accessions with specific traits, non-toxic, high yielding varieties). An in vitro germplasm collection of 1,200 accessions from 14 countries was established to (1) conserve valuable genetic resources, (2) survey genetic variation and (3) serve as starting material for genetic improvement with different breeding goals.

Low-Cost Methods for Molecular Characterization of Mutant Plants

This book offers low-cost and rapid molecular assays for the characterization of mutant plant germplasm. Detailed protocols are provided for the desiccation of plant tissues; the extraction of high-quality DNA for downstream applications; the extraction of single-strand-specific nucleases for single nucleotide polymorphism; and small insertion/deletion discovery using standard agarose gel electrophoresis. The methods described can be applied in any laboratory equipped for basic molecular biology and do away with the need for expensive freezers and toxic organic compounds. With the appropriate validation of sample quality and longevity, they can provide sufficient DNA for a variety of molecular applications, such as marker studies and TILLING, at approximately one tenth of the cost per sample when compared to commercial kits

Mutagenesis for Crop Breeding and Functional Genomics

Genetic variation is a source of phenotypic diversity and is a major driver of evolutionary diversification. Heritable variation was observed and used thousands of years ago in the domestication of plants and animals. The mechanisms that govern the inheritance of traits were later described by Mendel. In the early decades of the twentieth century, scientists showed that the relatively slow rate of natural mutation could be increased by several orders of magnitude by treating Drosophila and cereals with X-rays. What is striking about these achievements is that they came in advance of experimental evidence that DNA is the heritable material. This highlights one major advantage of induced mutations for crop breeding: prior knowledge of genes or gene function is not required to successfully create plants with improved traits and to release new varieties. Indeed, mutation induction has been an important tool for crop breeding since the release of the first mutant variety of tobacco in the 1930s. In addition to plant mutation breeding, induced mutations have been used extensively for functional genomics in model organisms and crops. Novel reverse-genetic strategies, such as Targeting Induced Local Lesions IN Genomes (TILLING), are being used for the production of stable genetic stocks of mutant plant populations such as Arabidopsis, barley, soybean, tomato and wheat. These can be kept for many years and screened repeatedly for different traits. Robust and efficient methods are required for the seamless integration of induced mutations in breeding and functional genomics studies. This chapter provides an overview of the principles and methodologies that underpin the set of protocols and guidelines for the use of induced mutations to improve crops.

Investigation of genetic variation in Jatropha curcas by Ecotilling and ISSR

Background The ability of species to adapt to different environments resides in their genetic diversity. This diversity, most commonly manifested as Single Nucleotide Polymorphisms (SNPs), can provide clues to the adaptive processes and population histories that have played a role in the species’ evolution. A number of different techniques for identifying SNPs have been developed, all having their limitations. Reverse genetics approaches rely on the detection of sequence alterations in target genes to identify allelic variations in natural or mutant populations. Ecotilling, a variant of TILLING (Targeting Induced Local Lesions IN Genomes) technique, allows high-throughput analyses of natural genetic diversity in plants [1], particularly in species with limited genetic diversity. Jatropha curcas L. is a perennial, monoecious shrub of the Euphorbiaceae family, native to America but distributed widely in the tropical and subtropical areas [2]. Wild or semi-cultivated types of J. curcas can grow well under unfavourable climatic and soil conditions [3]. J. curcas has attracted a great deal of attention worldwide, regarding its potential as a new energy plant. The seeds of J. curcas contain 30-45% oil [4] with a high percentage of monounsaturated oleic and polyunsaturated linoleic acid [5]. For genomic analyses, J. curcas is an interesting model species, since it has a relatively small genome (2C DNA content of 0.850 ± 0.006 pg or C DNA content of 0.416 × 109 bp) [6]. However, to achieve specific breeding goals in Jatropha for wider ecological adaptation, disease resistance and novel seed quality, the use of germplasm from different group and regions is necessary. Understanding the population structure of the alternative bioenergy plant Jatropha curcas is challenging due to limited genetic variability and information on phylogenetic relationships between accessions and related species. The development of cultivars of Jatropha curcas by conventional breeding will profit largely from biotechnological support (pathogen-free accessions with specific traits, non-toxic, high yielding varieties). The knowledge about J. curcas remains limited and little genomic research has been done so far [7]. In fact, the genetic map of J. curcas is not well-developed and only few molecular markers exist that could be used to clearly distinguish world wide accessions. Therefore, a resource database of SNPs in J. curcas would provide researchers with a tool for answering questions concerning population structure or adaptation and allow comparison of this species with related species.

Chemical Mutagenesis and Chimera Dissolution in Vegetatively Propagated Banana

Random mutagenesis has been widely used for forward-genetics and crop breeding since the application of ionising radiation on cereals described in the late 1920s. The development of high-throughput and accurate mutation discovery technologies has enabled reverse-genetic screening of mutant populations in the twenty-first century. The majority of mutation-based approaches in crops have involved seed-propagated species. Large bodies of data are available on the spectrum and density of induced mutations for some mutagens. It is well established that genetic chimerism caused by random accumulation of different mutations in different cells is resolved by sexual propagation and that by the second-generation post-mutagenesis (termed the M2), plants are no longer genetically mosaic. Vegetatively propagated species, however, are quite different as they primarily undergo mitotic propagation. In the absence of meiosis, procedures must be implemented to remove mosaicism and generate plant material that is genotypically homogeneous and suitable for forward- and reverse-genetic screening and breeding. We have previously developed a Targeting Induced Local Lesions IN Genomes (TILLING) platform for the vegetatively propagated triploid banana to investigate the density and spectrum of induced mutations and mechanisms by which tissue culture materials become genotypically homogeneous. Here we provide a detailed protocol for meristematic isolation, mutation induction and dissolution of chimeric sectors focusing on the use of chemical mutagen ethyl methanesulfonate (EMS).