Bradley J Till

Publications (31) View all

• Source

  Available from: Margit Laimer

  Article: Investigation of genetic variation in Jatropha curcas by Ecotilling and ISSR

  Fatemeh Maghuly, Joanna Jankowicz-Cieslak, Alberto Calari, Rose Ramkat, Bradley Till, Margit Laimer
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  ABSTRACT: The ability of species to adapt to different environments resides in their genetic diversity. This diversity, most commonly manifested as Single Nucleotide Polymorph-isms (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 ana-lyses of natural genetic diversity in plants [1], particu-larly in species with limited genetic diversity. Jatropha curcas L. is a perennial, monoecious shrub of the Euphorbiaceae family, native to America but distrib-uted 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 percen-tage 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 Jatro-pha for wider ecological adaptation, disease resistance and novel seed quality, the use of germplasm from dif-ferent 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 rela-tionships between accessions and related species. The development of cultivars of Jatropha curcas by conven-tional 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 lit-tle 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 concern-ing population structure or adaptation and allow com-parison of this species with related species. Methods The identification of novel SNPs that account for natural variation was used to study genetic diversity and the rela-tionships between and within Jatropha species. ISSRs (Inter Simple Sequence Repeats) also were considered as a tool in selecting germplasm for breeding purposes. An in vitro germplasm collection of 1300 accessions from 12 countries was established. This collection will serve different purposes: a) conserve valuable genetic resources, b) survey genetic variation, and c) serve as starting material for genetic improvement with different breeding goals. Ecotilling was applied to 12 different genes of interest related to stress tolerance, toxin and oil metabolism. 50 ISSR primers were used to assess the genetic diversity of Jatropha curcas and related species. Four different pool-ing strategies were used to identify homozygous and heterozygous SNP variations. In fact, variation was ana-lyzed both within a single tree (heterozygous) as well as between individual trees and a reference samples. Due to the reported low variations between Jatropha acces-sions [8,9] and large size of our collection, the 8 x 8 pooling strategy was chosen to estimate the level of var-iations among 12 selected genes. Results and conclusions To elucidate genetic relationship among Jatropha acces-sions from different regions and related species, a den-drogram was produced using NJ analysis of Nei´s genetic distance for 5 ISSR markers. The dendrogram is divided into two groups, one containing all Jatropha accessions and the other containing the related species. The main Jatropha curcas cluster is divided into two subclusters, one containing samples from Kenya and the other containing the remaining Jatropha accessions. The data showed clear variations not only among individuals but also between different regions. Ecotilling was found to be more efficient for large-scale studies of genetic variation in Jatropha, compared to RAPD, SSR and AFLP. Ecotilling is a low cost, high-throughput reverse genetic method for haplotyping and SNPs discovery.The level of differentiation observed was based on the geographic distribution pattern, i.e. it was higher in the centre of origin. ISSR analysis yielded highly reproducible patterns with 5/50 primers. Acknowledgements Financial support of FFG project 820132 and Bioplant R & D is acknowledged.
  BMC proceedings 07/2011; 26.
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  Available from: Bradley J Till

  Article: Discovery of nucleotide polymorphisms in the Musa gene pool by Ecotilling.

  Bradley J Till, Joanna Jankowicz-Cieslak, László Sági, Owen A Huynh, Hiroe Utsushi, Rony Swennen, Ryohei Terauchi, Chikelu Mba
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  ABSTRACT: 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.
  Theoretical and Applied Genetics 11/2010; 121(7):1381-9. · 3.30 Impact Factor
• Article: Optimizing TILLING and Ecotilling techniques for potato ( Solanum tuberosum L)

  Rana Elias, Bradley Till, Chikelu Mba, Bassam Al-Safadi
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  ABSTRACT: Abstract Background The TILLING and Ecotilling techniques for the discovery of nucleotide polymorphisms were applied to three potato ( Solanum tuberosum ) cultivars treated with gamma irradiation. The three mutant cultivars tested were previously shown to exhibit salinity tolerance, an important trait in countries like Syria where increasing soil salinity is affecting agricultural production. Findings Three gene-specific primer pairs were designed from BAC sequence to amplify ~1 to 1.5 kb of gene target. One of the three primer pairs amplified a single gene target. We used this primer pair to optimize enzymatic mismatch cleavage and fluorescence DNA detection for polymorphism discovery. We identified 15 putative nucleotide polymorphisms per kilobase. Nine discovered polymorphisms were unique to one of the three tetraploid cultivars tested. Conclusion This work shows the utility of enzymatic mismatch cleavage for TILLING and Ecotilling in different varieties of potato. The method allows for rapid germplasm characterization without the cost and high informatics load of DNA sequencing. It is also suitable for mutation discovery in high-throughput reverse genetic screens.
  BMC Research Notes. 01/2009;
• Source

  Available from: Kamila Kozak-Stankiewicz

  Dataset: 2011 Kozak et al. Inter-varietal differences

  Renata Galek, Kamila Kozak, Bado Souleymane, Ewa Sawicka-Sienkiewicz, Bradley J Till, Joanna Jankowicz-Cieslak
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  Available from: Bradley J Till

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

  Joanna Jankowicz-Cieslak, Owen A Huynh, Marta Brozynska, Joy Nakitandwe, Bradley J Till
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  ABSTRACT: 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.
  Plant Biotechnology Journal 08/2012; · 5.44 Impact Factor