Agrobacterium-mediated transfer of the Fusarium graminearum Tri6 gene into barley using mature seed-derived shoot tips as explants.

KEY MESSAGE: We transferred the Tri6 gene into the elite barley GemCraft via new transformation method through shoot organogenesis and identified the rearrangements of transgenes and phenotypic variations in the transgenic plants. Despite its agronomic and economic importance, barley transformation is still very challenging for many elite varieties. In this study, we used direct shoot organogenesis to transform the elite barley cultivar GemCraft with the RNAi constructs containing Tri6 gene of Fusarium graminearum, which causes fusarium head blight (FHB). We isolated 4432 shoot tips and co-cultured these explants with Agrobacterium tumefaciens. A total of 25 independent T0 transgenic plants were generated including 15 events for which transgene-specific PCR amplicons were observed. To further determine the presence of transgenes, the T1 progenies of all 15 T0 plants were analyzed, and the expected PCR products were obtained in 10 T1 lines. Droplet digital (dd) PCR analysis revealed various copy numbers of transgenes in the transgenic plants. We determined the insertion site of transgenes using long-read sequencing data and observed the rearrangements of transgenes. We found phenotypic variations in both T1 and T2 generation plants. FHB disease was evaluated under growth chamber conditions, but no significant differences in disease severity or deoxynivalenol accumulation were observed between two Tri6 transgenic lines and the wildtype. Our results demonstrate the feasibility of the shoot tip transformation and may open the door for applying this system for genetic improvement and gene function research in other barley genotypes.

QTL mapping of shoot and seed traits impacted by Drought in Barley using a recombinant inbred line Population.

BACKGROUND: With ongoing climate change, drought events are severely limiting barley production worldwide and pose a significant risk to the malting, brewing and food industry. The genetic diversity inherent in the barley germplasm offers an important resource to develop stress resiliency. The purpose of this study was to identify novel, stable, and adaptive Quantitative Trait Loci (QTL), and candidate genes associated with drought tolerance. A recombinant inbred line (RIL) population (n = 192) developed from a cross between the drought tolerant 'Otis' barley variety, and susceptible 'Golden Promise'(GP) was subjected to short-term progressive drought during heading in the biotron. This population was also evaluated under irrigated and rainfed conditions in the field for yields and seed protein content. RESULTS: Barley 50k iSelect SNP Array was used to genotype the RIL population to elucidate drought-adaptive QTL. Twenty-three QTL (eleven for seed weight, eight for shoot dry weight and four for protein content) were identified across several barley chromosomes. QTL analysis identified genomic regions on chromosome 2 and 5 H that appear to be stable across both environments and accounted for nearly 60% variation in shoot weight and 17.6% variation in seed protein content. QTL at approximately 29 Mbp on chromosome 2 H and 488 Mbp on chromosome 5 H are in very close proximity to ascorbate peroxidase (APX) and in the coding sequence of the Dirigent (DIR) gene, respectively. Both APX and DIR are well-known key players in abiotic stress tolerance in several plants. In the quest to identify key recombinants with improved tolerance to drought (like Otis) and good malting profiles (like GP), five drought tolerant RILs were selected for malt quality analysis. The selected drought tolerant RILs exhibited one or more traits that were outside the realms of the suggested limits for acceptable commercial malting quality. CONCLUSIONS: The candidate genes can be used for marker assisted selection and/or genetic manipulation to develop barley cultivars with improved tolerance to drought. RILs with genetic network reshuffling necessary to generate drought tolerance of Otis and favorable malting quality attributes of GP may be realized by screening a larger population.

Heat and drought induced transcriptomic changes in barley varieties with contrasting stress response phenotypes.

Drought and heat stress substantially impact plant growth and productivity. When subjected to drought or heat stress, plants exhibit reduction in growth resulting in yield losses. The occurrence of these two stresses together intensifies their negative effects. Unraveling the molecular changes in response to combined abiotic stress is essential to breed climate-resilient crops. In this study, transcriptome profiles were compared between stress-tolerant (Otis), and stress-sensitive (Golden Promise) barley genotypes subjected to drought, heat, and combined heat and drought stress for five days during heading stage. The major differences that emerged from the transcriptome analysis were the overall number of differentially expressed genes was relatively higher in Golden Promise (GP) compared to Otis. The differential expression of more than 900 transcription factors in GP and Otis may aid this transcriptional reprogramming in response to abiotic stress. Secondly, combined heat and water deficit stress results in a unique and massive transcriptomic response that cannot be predicted from individual stress responses. Enrichment analyses of gene ontology terms revealed unique and stress type-specific adjustments of gene expression. Weighted Gene Co-expression Network Analysis identified genes associated with RNA metabolism and Hsp70 chaperone components as hub genes that can be useful for engineering tolerance to multiple abiotic stresses. Comparison of the transcriptomes of unstressed Otis and GP plants identified several genes associated with biosynthesis of antioxidants and osmolytes were higher in the former that maybe providing innate tolerance capabilities to effectively combat hostile conditions. Lines with different repertoire of innate tolerance mechanisms can be effectively leveraged in breeding programs for developing climate-resilient barley varieties with superior end-use traits.

Deletion of the benzoxazinoid detoxification gene NAT1 in Fusarium graminearum reduces deoxynivalenol in spring wheat.

Benzoxazinoid (Bx) metabolites produced by wheat and other members of the Poaceae have activity against Fusarium sp. that cause cereal diseases including Fusarium head blight (FHB) on wheat and barley. Certain Bx metabolites can be detoxified by Fusarium sp. with the arylamine N-acetyltransferase NAT1. Investigation of this pathway may reveal strategies for increasing FHB resistance, such as selection for higher levels of Bx metabolites within existing germplasm and/or engineering fungal susceptibility via host induced silencing of NAT1. We assessed the reactions of fifteen wheat cultivars or breeding lines adapted to the Northwestern United States to infection with F. graminearum Δnat1 mutants that should be sensitive to Bx metabolites. Significant differences were noted in disease severity and deoxynivalenol (DON) among the cultivars 21 d after inoculation with either mutant or wildtype (PH1) strains. Mutant vs. wildtype strains did not result in significant variation for infection severity (as measured by % infected florets), but inoculation with Δnat1 mutants vs. wildtype resulted in significantly lower DON concentrations in mature kernels (p < 0.0001). Of the cultivars tested, HRS3419 was the most resistant cultivar to PH1 (severity = 62%, DON = 45 ppm) and Δnat1 mutants (severity = 61%, DON = 30 ppm). The cultivar most susceptible to infection was Kelse with PH1 (severity = 100%, DON = 292 ppm) and Δnat1 mutants (severity = 100%, DON = 158 ppm). We hypothesized that sub-lethal Bx metabolite levels may suppress DON production in F. graminearum Δnat1 mutants. In vitro assays of Bx metabolites BOA, MBOA, and DIMBOA at 30 µM did not affect growth, but did reduce DON production by Δnat1 and PH1. Although the levels of Bx metabolites are likely too low in the wheat cultivars we tested to suppress FHB, higher levels of Bx metabolites may contribute towards reductions in DON and FHB.

Impact on physiology and malting quality of barley exposed to heat, drought and their combination during different growth stages under controlled environment.

Drought and heat stress are two major abiotic stresses that tend to co-occur in nature. Recent climate change models predict that the frequency and duration of periods of high temperatures and moisture-deficits are on the rise and can be detrimental to crop production and hence a serious threat for global food security. In this study we examined the impact of short-term heat, drought and combined heat and drought stress on four barley varieties. These stresses were applied during vegetative stage or during heading stages. The impact on root and shoot biomass as well as seed yields were analyzed. This study demonstrated that sensitivity to combined stress was generally greater than heat or drought individually, and greater when imposed at heading than at the vegetative stages. Micromalted seeds collected from plants stressed during heading showed differences in malt extract, beta-glucan content and percent soluble protein. Screening barley germplasm during heading stage is recommended to identify novel sources of tolerance to combined stress. Apart from seed yield, assessing the seed quality traits of concern for the stakeholders and/or consumers should be an integral part of breeding programs for developing new barley varieties with improved heat and drought stress tolerance.

Silencing efficiency of dsRNA fragments targeting Fusarium graminearum TRI6 and patterns of small interfering RNA associated with reduced virulence and mycotoxin production.

Deoxynivalenol (DON) contamination of cereal grains caused by Fusarium head blight may be addressed by future RNA interference (RNAi)-based gene silencing approaches. However, utilizing these approaches will require a greater understanding of the principles that govern RNAi effectiveness in the pathogen Fusarium graminearum. RNAi in higher eukaryotes, including fungi, involves processing double stranded RNA (dsRNA) into small interfering RNA (siRNA) that silence gene expression based on base pair complementarity. This study examined virulence, DON production, and the small RNA (sRNA) populations in response to RNAi-based silencing of TRI6, a transcription factor that positively regulates DON synthesis via control of TRI5 expression. Silencing was accomplished via the expression of transgenes encoding inverted repeats targeting various regions of TRI6 (RNAi vectors). Transgene expression was associated with novel, TRI6-specific siRNAs. For RNAi vectors targeting the majority of TRI6 sequence (~600 bp), a discontinuous, repeatable pattern was observed in which most siRNAs mapped to specific regions of TRI6. Targeting shorter regions (250-350 bp) did not alter the siRNA populations corresponding to that region of TRI6. No phased processing was observed. The 5' base of ~83% of siRNAs was uracil, consistent with DICER processing and ARGONAUTE binding preferences for siRNA. Mutant lines showed TRI6 siRNA-associated reductions of TRI5 expression on toxin inducing media and DON in infected wheat and barley spikes. Shorter RNAi vectors resulted in variable levels of silencing that were less than for the ~600 bp RNAi vector, with a 343 bp RNAi vector targeting the 5' end of TRI6 having the best silencing efficiency. This work identifies efficient shorter region for silencing of TRI6 and describes the patterns of siRNA corresponding to those regions.

miR172 downregulates the translation of cleistogamy 1 in barley.

Background and Aims: Floret opening in barley is induced by the swelling of the lodicule, a trait under the control of the cleistogamy1 (cly1) gene. The product of cly1 is a member of the APETALA2 (AP2) transcription factor family, which inhibits lodicule development. A sequence polymorphism at the miR172 target site within cly1 has been associated with variation in lodicule development and hence with the cleistogamous phenotype. It was unclear whether miR172 actually functions in cly1 regulation and, if it does, which miR172 gene contributes to cleistogamy. It was also interesting to explore whether miR172-mediated cly1 regulation occurs at transcriptional level or at translational level. Methods: Deep sequencing of small RNA identified the miR172 sequences expressed in barley immature spikes. miR172 genes were confirmed by computational and expression analysis. miR172 and cly1 expression profiles were determined by in situ hybridization and quantitative expression analysis. Immunoblot analysis provided the CLY1 protein quantifications. Definitive evidence of the role of miR172 in cleistogamy was provided by a transposon Ds-induced mutant of Hv-miR172a. Key Results: A small RNA analysis of the immature barley spike revealed three isomers, miR172a, b and c, of which miR172a was the most abundant. In situ hybridization analysis showed that miR172 and cly1 co-localize in the lodicule primordium, suggesting that these two molecules potentially interact with one another. Immunoblot analysis showed that the sequence polymorphism at the miR172 target site within cly1 reduced the abundance of the CLY1 protein, but not that of its transcript. In a Ds-induced mutant of Hv-miR172a, which generates no mature miR172a, the lodicules fail to grow, resulting in a very small lodicule. Conclusions: Direct evidence is presented to show that miR172a acts to reduce the abundance of the CLY1 protein, which enables open flowering in barley.

Genome-wide analysis of the SPL/miR156 module and its interaction with the AP2/miR172 unit in barley.

The SQUAMOSA-promoter binding like (SPL) gene family encodes transcription factors that have been shown in many species to influence plant growth and development, but information about these genes in barley (Hordeum vulgare L.) is limited. This study identified 17 barley SPL genes, within eight distinct groups, that are orthologs of SPL genes described in Arabidopsis, wheat, and rice. Sixteen barley SPLs undergo alternative splicing. Seven SPLs contain a putative miR156 target site and the transcript levels of the miR156-targeted HvSPLs (HvSPL3, 13 and 23) were lower in vegetative than in reproductive phase but this was true also for some SPL genes such as HvSPL6 that were not regulated by miR156. Because SPL gene products regulate miR172, which is also involved in floral development, the expression of miR172 was studied. An antagonistic expression pattern of miR156 and miR172b during the vegetative and the reproductive phases signifies their apparent function in barley growth phase transition. Characterization of a barley mir172 mutant having an abnormal, indeterminate spikelet phenotype suggests the possible feedback role of AP2/miR172 module on HvSPL genes. This is the first comprehensive analysis of the miR156/SPL/miR172 axis in barley that provides a basis to elucidate their roles in various biological processes.

Sharing mutants and experimental information prepublication using FgMutantDb (https://scabusa.org/FgMutantDb).

There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species.

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.

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

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.

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.

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.

Developing tools for investigating the multiple roles of ethylene: identification and mapping genes for ethylene biosynthesis and reception in barley.

The plant hormone ethylene is important to many plant processes from germination through senescence, including responses to in vitro growth and plant regeneration. Knowledge of the number and function of genes that are involved in ethylene biosynthesis and reception is necessary to determine the role of specific genes within gene families known to influence ethylene biosynthesis and other aspects of ethylene function in plants. Our objective was built on previous studies that have established the critical role of ethylene in the in vitro response of barley (Hordeum vulgare L.), and that have identified ethylene-related QTL in the barley genome. In this study, we have identified the locations of genes in the barley 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO), and ethylene receptor (ETR) gene families. Specific primers for PCR amplification of each gene were developed and used to map these genes in the Oregon Wolf Barley mapping population. Five ACS, 8 ACO, and 7 ETR genes were identified and mapped to six of the barley chromosomes. Gene locations were syntenous to the orthologs in rice except for two that mapped to chromosome 6H. Gene duplication was evident for ACO genes on chromosomes 5H and 6H. Gene-specific primers will be useful for determining expression of each gene under various environmental conditions, including in vitro environments, to better understand the role of ethylene. Of the six known QTL for green plant regeneration in barley, three were located near the genes mapped in this study.

Compositional equivalence of barleys differing only in low- and normal-phytate levels.

Recent breeding advances have led to the development of several barley lines and cultivars with significant reductions (50% or greater) in phytate levels. Low-phytate (LP) grain is distinguished by containing not only a reduced level of phytate P but also an increased level of inorganic P, resulting in greater bioavailability of P and mineral cations in animal diets. It is important to determine whether other nutritional characteristics are altered by breeding for the low-phytate trait. This study was designed to investigate if breeding for reduced phytate content in barleys had any effect on the contents of other attributes measured by comparing mean and range values of the levels of protein, oil, ash, total carbohydrate, starch, and ß-glucan, fatty acid composition, and levels of tocopherols and tocotrienols between five LP and five normal-phytate barleys grown in three Idaho locations. Results show that only the phytate level in the LP group was substantially lower than that of the normal-phytate group and that all other attributes measured or calculated were substantially equivalent between the two groups of barleys. Therefore, the phytate level had little effect on the levels of protein, oil, ash, total carbohydrate, starch, and ß-glucan, fatty acid composition, and levels of tocopherols and tocotrienols in barley seeds.

Viability and bar expression are negatively correlated in Oregon Wolfe Barley Dominant hybrids.

The expression level of bar, which encodes phosphinothricin acetyltransferase (PAT), was correlated with the inviability of barley hybrids between 20 Golden Promise-derived transgenic lines (Ds-bar lines) and a specialized genetic marker stock, Oregon Wolfe Barley Dominant (OWBD). Each Ds-bar line was homozygous for a modified maize Ds element that encoded bar and that had been delivered via transposition to a unique location. All Ds-bar lines were viable and morphologically similar. Only four of the 20 hybrid populations were viable. The remaining populations died prior to producing seed. Phenotypic, enzyme-linked immunosorbent assay and quantitative reverse transcriptase-polymerase chain reaction analyses of these lines, and of lines from unrelated transformation events that also expressed bar, showed that viability was negatively correlated with bar expression. Analysis of crosses of a high-bar-expressing line with the OWB mapping population showed that the sensitivity of OWBD to PAT segregated as a single locus on chromosome 6HL. No sensitivity to PAT could be detected in several other lines and cultivars. OWBD has been shown to be genetically divergent from other germplasm groups within cultivated barley; therefore, the observed sensitivity may be peculiar to OWBD and thus would not impact generally on the utility of bar as a selectable marker or source of herbicide resistance in barley. Nevertheless, these results demonstrate the extent of allelic variability present in Hordeum vulgare, and suggest an additional variable for consideration when devising protocols for the transformation of Hordeum cultivars or landraces that are not known to be tolerant to PAT.

High-frequency Ds remobilization over multiple generations in barley facilitates gene tagging in large genome cereals.

Transposable elements have certain advantages over other approaches for identifying and determining gene function in large genome cereals. Different strategies have been used to exploit the maize Activator/dissociation (Ac/Ds) transposon system for functional genomics in heterologous species. Either large numbers of independent Ds insertion lines or transposants (TNPs) are generated and screened phenotypically, or smaller numbers of TNPs are produced, Ds locations mapped and remobilized for localized gene targeting. It is imperative to characterize key features of the system in order to utilize the latter strategy, which is more feasible in large genome cereals like barley and wheat. In barley, we generated greater than 100 single-copy Ds TNPs and determined remobilization frequencies of primary, secondary, and tertiary TNPs with intact terminal inverted repeats (TIRs); frequencies ranged from 11.8 to 17.1%. In 16% of TNPs that had damaged TIRs no transposition was detected among progeny of crosses using those TNPs as parental lines. In half of the greater than 100 TNP lines, the nature of flanking sequences and status of the 11 bp TIRs and 8-bp direct repeats were determined. BLAST searches using a gene prediction program revealed that 86% of TNP flanking sequences matched either known or putative genes, indicating preferential Ds insertion into genic regions, critical in large genome species. Observed remobilization frequencies of primary, secondary, tertiary, and quaternary TNPs, coupled with the tendency for localized Ds transposition, validates a saturation mutagenesis approach using Ds to tag and characterize genes linked to Ds in large genome cereals like barley and wheat.

Methylation of the exon/intron region in the Ubi1 promoter complex correlates with transgene silencing in barley.

Methylation of plant promoters is often associated with transcriptional gene silencing, while methylation of the transcribed region is associated with post-transcriptional gene silencing. Promoter complexes that include the first untranslated exon and intron, such as maize ubiquitin1 and rice actin1, have been widely used in monocot transformation because they support higher levels of transient transgene expression than the core promoter does. However, persistent problems with transgene silencing driven by these promoter complexes brought up a troubling question: were higher initial levels of transgene expression at the expense of long-term expression stability? Here we report that methylation of an untranslated exon and intron in the promoter complex is correlated with transcriptional transgene silencing in barley. Two sibling sublines, designated T(3)30 and T(3)31, were identified in a homozygous T3 population from a single transgenic parental line. In the T6 generation, all progeny of one subline, T(3)30, expressed ubiquitin-driven bar and uidA, but both transgenes were transcriptionally silenced in the other subline, T(3)31. Although the structure of the transgene locus is complex, no differences in the physical structure or location of the locus were detected between the two sublines. Transcriptional transgene silencing in T(3)31 correlated with two molecular events: methylation of the first untranslated exon and 5' end of the intron of the promoter complex, and condensation of the chromatin in regions containing transgenes. Passage of the non-silenced subline through in vitro culture recreated the silenced phenotype of T(3)31 and the molecular events leading to its silencing.