Low-Cost Methods for Molecular Characterization of Mutant Plants: Tissue Desiccation, DNA Extraction and Mutation Discovery: Protocols

Plant Breeding/Biotechnology; Biological Techniques; Nucleic Acid Chemistr

Low-Cost Methods for Molecular Characterization of Mutant Plants: Tissue Desiccation, DNA Extraction and Mutation Discovery: Protocols

Plant Breeding/Biotechnology; Biological Techniques; Nucleic Acid Chemistr

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 multidimensional 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

Discovery of nucleotide polymorphisms in the Musa gene pool by Ecotilling

Musa (banana and plantain) is an important genus for the global export market and in local markets where it provides staple food for approximately 400 million people. Hybridization and polyploidization of several (sub)species, combined with vegetative propagation and human selection have produced a complex genetic history. We describe the application of the Ecotilling method for the discovery and characterization of nucleotide polymorphisms in diploid and polyploid accessions of Musa. We discovered over 800 novel alleles in 80 accessions. Sequencing and band evaluation shows Ecotilling to be a robust and accurate platform for the discovery of polymorphisms in homologous and homeologous gene targets. In the process of validating the method, we identified two single nucleotide polymorphisms that may be deleterious for the function of a gene putatively important for phototropism. Evaluation of heterozygous polymorphism and haplotype blocks revealed a high level of nucleotide diversity in Musa accessions. We further applied a strategy for the simultaneous discovery of heterozygous and homozygous polymorphisms in diploid accessions to rapidly evaluate nucleotide diversity in accessions of the same genome type. This strategy can be used to develop hypotheses for inheritance patterns of nucleotide polymorphisms within and between genome types. We conclude that Ecotilling is suitable for diversity studies in Musa, that it can be considered for functional genomics studies and as tool in selecting germplasm for traditional and mutation breeding approaches

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

The effect of ionizing radiation on vernalization, growth and development of winter wheat

The effects of gamma irradiation on the vernalization requirements, growth and development of winter wheat grown in a rainout shelter were studied during two successive growing seasons. Dry grains of winter wheat cv. Kobra were irradiated with 300 Gy radiation from a cobalt 60 gamma irradiator. Treated and control grains were pregerminated and subjected to vernalization for 0, 42 or 54 days. Morphological parameters of the plants developing from irradiated seeds (M1 generation) and the plants grown from the seeds produced by the irradiated plants (M2 generation) were measured in order to track the studied effects over two generations. Irradiation of dry grains slowed the growth and development of the plants regardless of the temperature treatment. The measured yield structure elements appeared to be lower for irradiated plants, but no clear effect of radiation on vernalization requirements was noted