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.
KeywordsMutation breeding Reverse-genetics Forward-genetics Phenotyping Genotyping Technology packages
1.1 Inducing Genetic Variation
1.1.1 Practical Considerations in Induced Crop Mutagenesis
The choice of which type of mutagen to use for mutation breeding is often based on past successes reported for the species and other considerations such as the availability of mutagens, costs and infrastructure (Bado et al. 2015; Mba 2013; MVD 2016). Mutant varieties produced with ionising radiation, specifically gamma rays, predominate in the database of registered mutant varieties (MVD 2016). This may be due primarily to the active promotion of the use of gamma irradiation by the Food and Agriculture Organisation of the United Nations and the International Atomic Energy Agency (FAO/IAEA) Joint Programme, but also may be biologically significant as physical mutagens tend to induce larger genomic aberrations than some chemical mutagens, and more dominant or more easily observable traits could be created at a higher frequency (Jankowicz-Cieslak and Till 2015). Standardised protocols and general considerations for induced mutations in seed and vegetatively propagated plants using the physical mutagen (gamma rays) and the chemical mutagen (ethyl methanesulfonate, EMS) have been previously discussed (Lee et al. 2014; Bado et al. 2015; Till et al. 2006; Mba et al. 2010). Chapters 2, 3, 4 and 6 of this book describe chemical and/or physical mutagenesis protocols for obligate vegetatively propagated banana (Musa acuminata), facultative vegetatively propagated Jatropha (Jatropha curcas) and seed-propagated barley (Hordeum vulgare).
A major bottleneck in plant mutation breeding is the imperative of generating and evaluating large mutant populations in order to increase the chance of identifying a desirable variant. Efforts are devoted to the dissociation of chimeras, also known as mosaics or sectoral differences, whereby cells of different genotypes exist side by side in the tissues of the same mutant plant. This is straightforward in sexually produced crops owing to the fact that single cells in the form of gametes are the basis for the next generation, thus resolving any chimeras. For vegetatively propagated crops, several cycles of regeneration may be required to produce solid homohistonts or genotypically homogeneous material (van Harten 1998; Mba et al. 2009). One way to avoid chimerism in vegetatively propagated species is to mutagenise individual cells that can regenerate into plants, either using cell suspensions or (embryogenic) callus (van Harten 1998). Protocols for these strategies are provided in Chaps. 4 and 5. These approaches have been less often used than those involving multicellular organs and tissues, and so there is less information available on the possibility of chimerism at the DNA sequence. It is interesting to speculate on the fate of induced DNA modifications in single cells. For example, EMS mutagenesis results in alkylation, whereby the original base is not physically altered, but the mutation is only fixed due to an error in replication of the affected base. Here, two daughter cells could be produced with distinct genotypes.
1.1.2 Developing Crop Varieties Using Induced Mutations
1.1.3 Elite Crop Varieties Developed Through Induced Mutations
Ahloowalia et al. (2004) and Kharkwal and Shu (2009) provided overviews of the contributions of these mutant crop varieties to food security and nutrition and economic wellbeing. Widely cultivated rice varieties in Australia, China, India, Pakistan and Thailand; sunflower and peppermint in the USA; barley in many countries of Europe; sorghum in Mali; and several ornamental plants in India, the Netherlands and Germany are a few such examples. The high-yielding and dwarf mutant cultivars of barley, ‘Diamant’ and ‘Golden Promise’, for instance, and their progenies are credited with additional billions of dollars in revenues for the brewing and malting industries in Europe (Ahloowalia et al. 2004). Other examples include most of the varieties of durum wheat grown in Italy for pasta and marketed worldwide; the Rio Star grapefruit in the USA; the Japanese pear variety, ‘Gold Nijesseiki’; and the cotton variety, NIAB78, in Pakistan.
It is difficult to estimate the precise extent to which novel alleles created through mutagenesis have been used in developing superior crop varieties worldwide. From a practical standpoint in most parts of the world, they are treated like any allele that the breeder may knowingly or unknowingly incorporate into a new improved cultivar.
1.2 Phenotypic Screening
The origin of agriculture can be traced back to more than 10,000 years ago when the first crops were domesticated in the Fertile Crescent (Brown et al. 2009). The identification (phenotyping) and deliberate selection of off-type plants (i.e. spontaneous mutants) by the then hunters and gatherers were the initial efforts at plant breeding. The first plants to undergo domestication are thought to be the cereals wheat, barley, millet and emmer (Sang 2009). Early phenotypers selected plants with increased grain size and loss of seed shattering. They created and improved crops without any knowledge of why the selected differences occurred or were heritable. The first written reports of the earliest description of the selection of (presumably) spontaneous mutants appear to date back to around 300 BC when selection of plants with abnormal but improved traits were described in an ancient Chinese book, ‘Lulan’ (Shu et al. 2012). Among the improved characters were ‘days to maturity’ and other easily visible traits in cereal crops. The term ‘mutation’ was coined much later by Hugo de Vries to describe sudden genetic change in higher plants which was stably inherited through many years (de Vries 1901). Spontaneous mutants remain valuable sources of diversity and variation, but many years of intensive breeding has resulted in the narrowing of the genetic base of many crop species necessitating the need to create new variation through means such as induced mutations.
Accurate plant phenotyping remains as critically important today as it has ever been and is arguably the major bottleneck in plant breeding (Fiorani and Schurr 2013). This is especially true in plant mutation breeding where a novel trait may appear only once in a population of several thousand mutant lines. Therefore, high-throughput and cost-efficient screening methods are required for the rapid identification and characterisation of putative mutants.
1.2.1 Phenotypic Traits Developed Through Plant Mutation Breeding
Examples of released improved varieties registered in the Mutant Variety Database under five main trait categories
Mutant’s name (species)
Agronomic and botanic traits
‘Above’ (Triticum aestivum L.)
Awned, white glumed, early maturing and semidwarf
Treatment of seed with chemical mutagen, sodium azide (NaN3)
Newhouse et al. (1992)
Quality and nutrition traits
‘Aldamla’ (Prunus avium L.)
Compact growth habit (70–80 %), long petioles and improved fruit quality
Irradiation of dormant buds with gamma rays
Kunter et al. (2012)
Resistance to biotic stresses
‘Akita Berry’ (Fragaria x ananassa)
Improved resistance to black leaf spot disease (Alternaria alternata)
Somaclonal mutation by meristem culture
Tolerance to abiotic stresses
‘Maybel’ (Lycopersicon esculentum M.)
Very high performance under drought conditions
Treatment of seed with gamma rays
Yield and contributors
‘Early Blenheim’ (Prunus armeniaca L.)
Early maturity, higher yield, large fruits and self-compatible pollen
Treatment of dormant scions with thermal neutrons (thN)
Sigurbjoernsson and Micke (1974)
Interestingly, the majority (48 %) of released mutant varieties that are registered in the Mutant Variety Database are characterised by improved agronomic and botanic traits. This could be due to the fact that botanic and agronomic traits are easily observable, and for most of them, no specialised equipment is needed for screening. The least represented mutants are in the biotic and abiotic stresses category. It is notable that these characters, though complex and difficult to screen for, are important breeding objectives. Examples of officially released mutant varieties in the five trait categories are listed in Table 1.1.
There remains a clear need to develop methods and protocols to enhance the efficiency of the mutation breeding process. The protocols in this book aim to do this, but it remains an incredibly challenging task. Every step of the procedure can differ depending on the parental genotype, propagation mode (seed versus vegetatively), the trait of interest which needs to be improved and available facilities. For example, Near-Infrared Reflectance Spectroscopy (NIRS) is a method that can be used to screen for seed composition. Traditional methods used a destructive approach which is suitable for characterising an advanced mutant line where many seeds are available. Screening is rapid, and non-destructive methods that measure whole seed allow NIRS to be used as a fast prescreen of large mutant populations. Protocols for both destructive and non-destructive screening of rice are provided in Chap. 12. Calibration standards can be applied to NIRS spectra to evaluate seed components such as protein content. Once interesting mutants are identified in a rapid prescreen, detailed characterisation can be undertaken. Proteomic analysis allows a detailed cataloguing of the effect of genetic variation on the collection of expressed proteins in grains or tissues. Chapter 13 of this book provides detailed methods for protein analysis in seed and leaves of Jatropha curcas. Digital imaging is another non-destructive method that can be adapted for phenotypic evaluation of morphological variations induced by treatment with mutagens. Root architecture, for example, is an important component in abiotic stress responses such as drought. A low-cost approach for digital analysis of root traits is provided in Chap. 10.
1.3 Genotypic Screening of Mutant Plants
1.3.1 Genotypic Methods
Plant genotyping can be broadly considered as any experimental assay that aims to evaluate differences in the nucleotide sequence within or between species. This is an especially powerful approach because nucleotide variation is the major contributor to heritable phenotypic variation. Methods to uncover nucleotide variation also provide important information on plant evolution and enable efficient selections that avoid the confounding effects of genotype by environment (GxE) interactions (Annicchiarico 2002). Protocols for genomic DNA acquisition and evaluation have been improving to the point where resequencing of hundreds to thousands of plant genomes is now a reality (Weigel and Mott 2009).
126.96.36.199 Lower-Cost Mutation Discovery and Genotyping Methods
The risk of new technologies, however, is that they tend to be expensive and require a high level of technical expertise. New tools, therefore, are not available to all laboratories. Yet many powerful methods can be developed that are lower cost and suitable for laboratories with varying infrastructure. One example is the starting point of all genotyping experiments: the extraction of DNA. While long-term storage of plant tissues prior to DNA extraction often involves the use of liquid nitrogen and −80 °C freezers, these can be avoided by desiccating and storing leaf material in silica gel at room temperature (Till et al. 2015). Extraction of high-quality genomic DNA from leaf material is typically performed using expensive kits or with more manual methods that require toxic organic chemicals such as the CTAB method. These can be avoided by using the protocol described in Chap. 14 of this book. DNA is extracted by binding to silica in the presence of chaotropic salts. This mirrors the chemistry used in expensive kits but at about only 10 % of the price. Importantly, therefore, high-quality genomic DNA can be extracted without specialised equipment for tissue grinding and without the use of any toxic organic compounds that require specialised waste disposal.
Low-cost methods do not end with the extraction of genomic DNA. The process of altering the expression or activity of a gene in order to evaluate its function in vivo is known as reverse-genetics. This term was coined because it is essentially the reverse of the process of forward-genetics which starts with a phenotype and ends with a gene sequence. While endogenous transposons have been used for gene disruptions in some crops such as maize and rice, a major development came in the late 1990s with a reverse genetic approach known as TILLING that uses induced mutations (Meeley and Briggs 1995; McCallum et al. 2000; Hirochika 2001; Conrad et al. 2008; Hunter et al. 2014). TILLING, short for Targeting Induced Local Lesions IN Genomes, typically utilises mutagens that induce a high density of induced mutations randomly throughout the genome (Kurowska et al. 2011; Greene et al. 2003; Jankowicz-Cieslak et al. 2011). A population of between 3000 and 6000 mutant lines can be developed that contains multiple mutations in every gene in the genome. A library of DNA and seed can be prepared and used as a resource for many years. In traditional TILLING, the DNA library is screened by PCR and enzymatic mismatch cleavage to identify mutations in target genes of choice. The entire TILLING process can be made low cost. In addition to low-cost methods for DNA extraction found in Chap. 14, Chap. 15 describes a rapid method for the extraction of single-strand-specific nucleases for TILLING and other applications that costs less than 1 cent per assay. Standard agarose gels can be used as a readout platform for mutation discovery.
188.8.131.52 Higher-Throughput Genotyping and Mutation Discovery Methods
Where budgets permit, next-generation sequencing technologies offer significant gains in screening throughput over low-cost methods. For example, the TILLING by sequencing protocol described in Chap. 20 provides a three-dimensional pooling strategy for 768 individuals and simultaneous mutation discovery in many gene targets (Tsai et al. 2011). The use of advanced tools is not limited to reverse-genetics. The majority of officially released mutant crop varieties are produced from forward-genetic screens from plant materials treated with ionising radiation (MVD 2016). Genomic techniques promise to greatly enhance the efficiency of traditional forward mutation breeding that has been a mainstay for over 70 years. The challenge remains to determine if a population truly harbours a high density of desirable mutations. While visual evaluation of M1 plants as described above is advantageous in that it is rapid and low-cost, it is known that variations observed in the M1 do not represent heritable DNA mutations (Preuss and Britt 2003). Therefore, mutation density need not correlate with phenotypic variations observable in the M1. Next-generation sequencing technologies now provide rapid methods for the evaluation of mutation density and spectra in the M2 generation. Many plant genomes are prohibitively large to consider whole-genome sequencing of the requisite number of plants for all but the very highly funded laboratories. Reduced representation genome sequencing offers a solution. Chapter 19 provides a protocol for Restriction Enzyme Sequence Comparative Analysis (RESCAN) where a fraction of a plant genome can be sequenced for discovery of induced point mutations. Here, tens of millions of base pairs can be sequenced from each mutant plant to recover sufficient mutations for a suitable estimation of mutation density.
184.108.40.206 Cloning Mutant Alleles Causative for Improved Traits
The same protocols described above can be adapted for another major challenge of forward mutation breeding: the identification and cloning of mutations causing the improved trait. With smaller genome plants, it is possible to sequence whole genomes and clone genes by associating co-segregation of genotype to phenotype (Schneeberger et al. 2009; Cuperus et al. 2010). An approach known as MutMap has been described for cloning EMS-induced alleles in rice using a bulked segregant strategy, and the method further adapted so that alleles can be cloned without outcrossing (Abe et al. 2012; Fekih et al. 2013). This is much more challenging in larger genome crops due to throughput and cost limitations of whole-genome sequencing. Targeted capture-resequencing methods offer a way for reduced representation genome sequencing of specific regions designed by the researcher. Coding sequences are an excellent choice when mutations affecting gene function are sought. Henry and colleagues describe exome capture methods to recover EMS-induced mutations in rice and wheat (Henry et al. 2014). With large genomes like wheat, this approach allows massive enrichment of functional regions of the genome and makes applications such as MutMap feasible, so long as causative mutations lie within regions that the researcher has selected for sequencing. To date, the majority of efforts have focused on recovery of point mutations such as those induced by treatment with the chemical mutagen EMS. Less is known about the effects of mutagens such as gamma irradiation, but recent experiments suggest that mutagen causes primarily large genomic deletions. For example, deletions of 1.2 million base pairs and 232,000 base pairs were recovered in Zea mays treated with gamma irradiation (Yuan et al. 2014). In sorghum, deletions ranging between 100,000 kb and 700,000 kb were recovered in materials treated with 75 and 300 Gy (B.J Till, I.M. Henry and L. Comai, unpublished). In contrast to what is emerging from gamma irradiation, whole-genome sequencing studies of fast neutron-irradiated rice suggest a broader spectrum of mutations (Li et al. 2016).
The presence of large genomic deletions may make the task of cloning much easier. For example, a diploid such as sorghum with a ~ 730 Mbp genome that is treated with EMS may harbour 3000 induced mutations making the job of finding the one mutation causing the trait difficult. The same genome treated with gamma irradiation may only be able to accumulate a small number of large genomic indels. Thus, identifying the mutation causing the phenotype is severalfold easier. Discovery of large genomic indels via sequencing may also prove more efficient as lower depth of coverage is needed for accurate variant calling compared to SNPs, and therefore more samples can be screened per run. This approach has been used to catalogue gamma-induced mutations created through irradiation of pollen from poplar (Henry et al. 2015). Continued improvement in sequencing technologies suggests that cloning both SNP and large indel mutations will become more common in the near future. This will make valuable mutant alleles available to breeders for marker-assisted introgression into elite germplasm.
There is little controversy that growing pressures on agricultural productivity such as increasing population, reduction of arable land and new and geographically shifting biotic and abiotic stresses demand serious attention and innovative approaches. Genetic improvement of crops is fundamental to long-term success, and a combination of novel developments and translational science is required. We predict that induced mutagenesis will remain an important tool for the breeder as it is a rapid and relatively low-cost approach to generate novel alleles and phenotypes. Further, new technologies will enable determination of mutant alleles used to create successful mutant varieties and will shed light on gene function and crop productivity.
Funding for this work was provided by the Food and Agriculture Organization of the United Nations and the International Atomic Energy Agency through their Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture. This work is part of IAEA Coordinated Research Project D24012.
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