Phil Bregitzer

Genetic basis and detection of unintended effects in genetically modified crop plants

In January 2014, an international meeting sponsored by the International Life Sciences Institute/Health and Environmental Sciences Institute and the Canadian Food Inspection Agency titled “Genetic Basis of Unintended Effects in Modified Plants” was held in Ottawa, Canada, bringing together over 75 scientists from academia, government, and the agro-biotech industry. The objectives of the meeting were to explore current knowledge and identify areas requiring further study on unintended effects in plants and to discuss how this information can inform and improve genetically modified (GM) crop risk assessments. The meeting featured presentations on the molecular basis of plant genome variability in general, unintended changes at the molecular and phenotypic levels, and the development and use of hypothesis-driven evaluations of unintended effects in assessing conventional and GM crops. The development and role of emerging “omics” technologies in the assessment of unintended effects was also discussed. Several themes recurred in a number of talks; for example, a common observation was that no system for genetic modification, including conventional methods of plant breeding, is without unintended effects. Another common observation was that “unintended” does not necessarily mean “harmful”. This paper summarizes key points from the information presented at the meeting to provide readers with current viewpoints on these topics.

Mapping Ds insertions in barley using a sequence-based approach

A transposon tagging system, based upon maize Ac/Ds elements, was developed in barley (Hordeum vulgare subsp. vulgare). The long-term objective of this project is to identify a set of lines with Ds insertions dispersed throughout the genome as a comprehensive tool for gene discovery and reverse genetics. AcTPase and Ds-bar elements were introduced into immature embryos of Golden Promise by biolistic transformation. Subsequent transposition and segregation of Ds away from AcTPase and the original site of integration resulted in new lines, each containing a stabilized Ds element in a new location. The sequence of the genomic DNA flanking the Ds elements was obtained by inverse PCR and TAIL-PCR. Using a sequence-based mapping strategy, we determined the genome locations of the Ds insertions in 19 independent lines using primarily restriction digest-based assays of PCR-amplified single nucleotide polymorphisms and PCR-based assays of insertions or deletions.The proncipal strategy was to identify and map sequence polymorphisms in the regions corresponding to the flanking DNA using the Oregon Wolfe Barley mapping population. The mapping results obtained by the sequence-based approach were confirmed by RFLP analyses in four of the lines. In addition, cloned DNA sequences corresponding to the flanking DNA were used to assign map locations to Morex-derived genomic BAC library inserts, thus integrating genetic and physical maps of barley. BLAST search results indicate that the majority of the transposed Ds elements are found within predicted or known coding sequences. Transposon tagging in barley using Ac/Ds thus promises to provide a useful tool for studies on the functional genomics of the Triticeae.

Plant regeneration from barley callus: Effects of 2,4-dichlorophenoxyacetic acid and phenylacetic acid

The use of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-d) has played an important role in the production and maintenance of totipotent cereal callus. However, 2,4-d has been implicated in the loss of totipotency from barley callus. To examine the effect of 2,4-d on barley callus, regenerability and karyotype were examined over time as influenced by cultivar differences and 2,4-d levels, during a period in which initially vigorous plant regeneration typically declines dramatically. Higher (20.4–27.1 μM) versus lower (6.8–13.6 μM) concentrations of 2,4-d were positively associated with the number of green plantlets recovered from calli maintained for 10 and 16 weeks before transfer to regeneration media, and with the longevity of regenerability. There was a positive relationship between 2,4-d concentration and normal karyotype. We also investigated the use of phenylacetic acid for the initiation of regenerable barley callus. Very poor callus growth and plant regeneration was supported by phenylacetic acid.

Transgenic Cereals: Hordeum vulgare L. (barley)

The development of barley as a crop dates to the earliest agricultural activities of humans, and it remains one of the major cereals grown for feed and food, and for the production of beer. In this century, an understanding and application of quantitative genetic theory has created a genetically elite crop that is divergent from its ancestors. Further improvements in barley cultivare will depend on continued access to useful allelic variability. Sexual hybridization will continue to play an important role in such improvement, but its utility is limited because potentially useful alleles are either linked to undesirable alleles or unavailable because of sexual incompatibilty. The advent of molecular genetics and nonsexual gene transfer offers exciting opportunities to bypass these limitations and to provide access to more diverse sources of genes. Recent developments have added barley to the list of major crops that are amenable to this type of genetic manipulation either through direct DNA transfer (bombardment) or mediated by Agrobacterium tumefaciens. However, significant problems remain, and include: 1) the lack of reproducible, efficient transformation systems for commercial germplasm; 2) the induction of stable genetic and epigenetic changes during the in vitro process; and 3) transgene and transgene expression instability. In this chapter, we will discuss and describe the first systems used for the genetic transformation of barley, introduce and describe the development of new systems for barley transformation, and comment on past and future uses of barley transformation as a tool for basic science and commercial application.

Sequencing of 15 622 gene-bearing BACs clarifies the gene-dense regions of the barley genome

Summary Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole‐genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene‐containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical‐mapped gene‐bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene‐enriched BACs and are characterized by high recombination rates, there are also gene‐dense regions with suppressed recombination. We made use of published map‐anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D‐genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley–Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map‐based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene‐dense but low recombination is particularly relevant.

Effects of Four Independent Low-Phytate Mutations in Barley (Hordeum vulgare L.) on Seed Phosphorus Characteristics and Malting Quality

ABSTRACT Conversion of the seed phosphorus storage compound phytic acid, which is poorly digested by nonruminants, to available forms of phosphorus will have nutritional and environmental benefits. Low-phytate (LP) barley (Hordeum vulgare L.) cultivars are in development and their commercialization will be facilitated by understanding their phosphorus profiles and malting quality. To study these issues, LP and normal types derived from mutagenized populations of barley cultivar Harrington (sets of sib lines homozygous for the wild-type [WT] allele, or for one of four low-phytic acid mutations, lpa1-1, lpa2-1, lpa3-1, or M955), were developed through backcrosses to Harrington. Grain was produced in irrigated and rain-fed environments. WT phosphorus profiles were similar to those of Harrington, suggesting that the major variable was the presence or absence of mutant alleles. All mutations conferred increased inorganic phosphorus. Total P was reduced for lpa1-1. Phosphorus profiles were relatively stable acr...

Comparative analysis of genomic DNA methylation status and field performance of plants derived from embryogenic calli and shoot meristematic cultures

Genomic instability has been widely observed in in vitro-derived plants; it is proposed to result from the break-down of normal cellular control (Phillips et al, 1994). Following DNA introduction into scutella directly or embryogenic callus derived from the scutella of immature embryos (IEs), numerous transgenic barley lines were produced (Wan and Lemaux, 1994; Lemaux et al, 1996). Evaluation of these lines revealed severe reduction in agronomic performance in field tests (Bregitzer et al., 1998) and instability of the transgene and its expression (Bregitzer et al., unpublished data). We propose that poor performance may be in part due to in vitro-induced genetic or epigenetic variability. Plants derived from alternative culture methods were analyzed to study the effects of these methods on genomic stability. Plants were regenerated from tissue obtained using methods of standard embryogenic culture (Wan and Lemaux 1994), modified embryogenic callus (Cho et al., 1998) and cultured shoot meristems. Initial results with plants derived from the various methods will be compared relative to methylation polymorphism and field performance.

A High-Throughput RNA Extraction for Sprouted Single-Seed Barley (Hordeum vulgare L.) Rich in Polysaccharides

Germinated seed from cereal crops including barley (Hordeum vulgare L.) is an important tissue to extract RNA and analyze expression levels of genes that control aspects of germination. These tissues are rich in polysaccharides and most methods for RNA extraction are not suitable to handle the excess polysaccharides. Here, we compare the current methods for RNA extraction applicable to germinated barley tissue. We found that although some of these standard methods produced high-quality RNA, the process of extraction was drastically slow, mostly because the frozen seed tissue powder from liquid N2 grinding became recalcitrant to buffer mixing. Our suggested modifications to the protocols removed the need for liquid N2 grinding and significantly increased the output efficiency of RNA extraction. Our modified protocol has applications in other cereal tissues rich in polysaccharides, including oat.

Long-term assessment of transgene behavior in barley: Ds-mediated delivery of bar results in robust, stable, and heritable expression

The utility of transgenic plants for both experimental and practical agronomic purposes is highly dependent on stable, predictable, and heritable expression of the introduced genes. This requirement is frequently unfulfilled, and transgenes often are completely silenced. Studies of transgenic loci have shown that rearrangements of transgenes occur during the integration process, some of which are potent cues that induce silencing. Conversely, intact, single-copy transgenes produced via transposon-mediated gene delivery have shown relatively stable expression, at least in early-generation progeny. To examine the long-term expression stability of a bar expression cassette delivered via Dissociation (Ds)-mediated transposition, we examined qualitative and quantitative expression in barley (Hordeum vulgare L.) populations developed for transposon tagging. Qualitative assessments of herbicide resistance among 106 lines showed bar expression to be stable for at least five generations of advance via self-pollination. Similarly, qualitatively stable expression was observed among 31 near-isogenic lines derived from at least seven backcrosses to the cultivar Garnet. Quantitative RT-PCR measurements of bar expression were conducted for eight near-isogenic lines and their donor parents. The expression of bar was highly correlated in parent and progeny near-isogenic lines, showing high heritability of bar expression. These data demonstrate stable, predictable transgene expression following Ds-mediated delivery.

A Substantial Fraction of Barley (Hordeum vulgare L.) Low Phytic Acid Mutations Have Little or No Effect on Yield across Diverse Production Environments

The potential benefits of the low phytic acid (lpa) seed trait for human and animal nutrition, and for phosphorus management in non-ruminant animal production, are well documented. However, in many cases the lpa trait is associated with impaired seed or plant performance, resulting in reduced yield. This has given rise to the perception that the lpa trait is tightly correlated with reduced yield in diverse crop species. Here we report a powerful test of this correlation. We measured grain yield in lines homozygous for each of six barley (Hordeum vulgare L.) lpa mutations that greatly differ in their seed phytic acid levels. Performance comparisons were between sibling wild-type and mutant lines obtained following backcrossing, and across two years in five Idaho (USA) locations that greatly differ in crop yield potential. We found that one lpa mutation (Hvlpa1-1) had no detectable effect on yield and a second (Hvlpa4-1) resulted in yield losses of only 3.5%, across all locations. When comparing yields in three relatively non-stressful production environments, at least three lpa mutations (Hvlpa1-1, Hvlpa3-1, and Hvlpa4-1) typically had yields similar to or within 5% of the wild-type sibling isoline. Therefore in the case of barley, lpa mutations can be readily identified that when simply incorporated into a cultivar result in adequately performing lines, even with no additional breeding for performance within the lpa line. In conclusion, while some barley lpa mutations do impact field performance, a substantial fraction appears to have little or no effect on yield.