Deletion Mutagenesis Identifies a Haploinsufficient Role for γ-Zein in opaque2 Endosperm Modification

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.

Section: Cloning Mutant Alleles Causative For Improved Traitsmentioning

confidence: 99%

Mutagenesis for Crop Breeding and Functional Genomics

Jankowicz-Cieślak

Chikelu

Till

2016

“…For example, a mutation density of~1/280 kb in rice has been reported (Till et al 2007;Tsai et al 2011). Physical mutagens such as gamma irradiation have been shown to produce large genomic indels with only a few such lesions reported per line (Yuan et al 2014;Henry et al 2015). This means that the chance of mutating a gene that would alter the desired trait is quite low, and thousands of plants need to be routinely screened for a chance of success.…”

Section: Introductionmentioning

confidence: 99%

Field Evaluation of Mutagenized Rice Material

Johnson

Taylor

Tai

et al. 2016

Since the discoveries of the beneficial effects of some mutations on plants, scientists have made use of both physical and chemical mutagens to develop new varieties of crops and ornamentals. More than 3200 varieties have been produced through mutation induction, and these have contributed towards improving food security in many countries.Proper field techniques can greatly enhance the chances of identifying desirable phenotypes. The protocol described here deals mainly with field techniques successfully employed and developed in the evaluation of rice mutants for over 8 years in the uplands and the hydromorphic lowlands at the Rokupr Agricultural Research Centre (RARC) of the Sierra Leone Agricultural Research Institute (SLARI) between 2005 and 2012 to evaluate gamma and X-ray-treated rice seeds.

“…In rice, data suggest that >80 % of mutations induced via gamma irradiation are small deletions up to 16 base pairs, with the remainder being large deletions (Sato et al 2006;Bruce et al 2009). Yet in maize only deletions greater than 200 kilobases were reported (Yuan et al 2014). Some caution is needed when trying to draw broad conclusions from these datasets as many technologies employed to discover mutations have strong ascertainment biases.…”

Section: Introductionmentioning

confidence: 99%

A Protocol for Benchtop Extraction of Single-Strand-Specific Nucleases for Mutation Discovery

Hofinger

Huynh

Jankowicz-Cieślak

et al. 2016

Single-strand-specific nucleases are used to cleave mismatches in otherwise double-stranded DNA. Assays typically involve PCR amplification followed by a denaturing and annealing step to generate heteroduplexed DNA molecules from PCR products containing nucleotide polymorphisms. This is followed by digestion with nucleases that cleave at the site of the mismatch. The molecular weights of the cleavage products indicate the approximate position of nucleotide polymorphisms in PCR amplicons. Cleaved DNA products are observed by one of several readout platforms such as native gel electrophoresis, denaturing gel electrophoresis, capillary electrophoresis, or denaturing high-performance liquid chromatography (DHPLC). This approach is highly suitable for accurate discovery of natural and induced single-nucleotide polymorphisms (SNPs) and also small insertions and deletions (indels). The use of self-extracted single-strand-specific nucleases, typically prepared from crude extracts of celery (Apium graveolens), is common for reverse-genetics (e.g. Targeting Induced Local Lesions IN Genomes (TILLING)) and studies of natural nucleotide polymorphisms (e.g. Ecotilling). While protocols have been published describing the preparation of single-strandspecific nuclease from plant tissues, many rely on toxic chemicals, dialysis and large-volume preparatory centrifuges and also require steps to be performed at 4 C. We provide here a streamlined room temperature extraction protocol that uses standard microcentrifuges and eliminates toxic chemicals and traditional dialysis.