Barley is a major grain crop in Canada and is used for generating malt, in addition to being used as human food and animal feed. Differentiating barley varieties is important for malt quality assurance and grain handling. Here, we present a DNA-based testing method for the identification of Canadian barley varieties. The method uses 24 custom TaqMan genotyping assays, which are analyzed using the high-throughput SmartChip system (Takara Bio Inc.). Using this method, we are able to distinguish 124 barley varieties commonly grown in Canada.

In this study, we report an updated panel of 32 DNA markers used for identification of wheat varieties and assess their performance in the OpenArray and SmartChip high-throughput genotyping systems. While both systems are unique and offer different advantages and disadvantages, both systems can successfully identify Canadian wheat varieties.

We developed a set of 16 markers in durum wheat for use on the TaqMan OpenArray Genotyping system. When used in combination with automated variety calling, it provides a simple and efficient means to identify Canadian durum varieties and assess variety composition of seed and (or) grain samples.

Analysis of bulk ground samples by droplet digital PCR (ddPCR) was investigated as an alternative to individual kernel testing for assessment of interspersed refuge in midge [Sitodiplosis mosellana (Gehin)] tolerant wheat (Triticum aestivum L.) varietal blends. Four genotyping assays were selected such that at least one assay was informative for each of 15 varietal blends registered for production in Canada. The assays were examined in DNA of each of the constituent varieties to assess intravarietal polymorphism and in DNA mixtures simulating each varietal blend. Seed mixtures corresponding to a range of refuge proportions in two different varietal blends were prepared and assessed by ddPCR on two different platforms (RainDrop Digital PCR System, RainDance Technologies, Billerica, MA, and QX200 AutoDG Droplet Digital PCR System, Bio-Rad Laboratories, Hercules, CA). Both systems yielded refuge estimates that were very close to the targeted proportions across the range simulated. Standard deviations among estimates made with the QX200 were, on average, about 60% greater than those among corresponding estimates made with the RainDrop system, but in either case, the coefficients of variation generally remained under 5%. For a ddPCR-based assessment of interspersed refuge in midge tolerant wheat varietal blends, any advantage held by the RainDrop system with respect to precision may be offset by higher throughput achievable with the QX200.

A multiplexed set of SSR markers was assembled which, in combination with DNA extraction from individual seeds, provides a practical means to identify Canadian flax varieties and may be useful for assessment of seed purity. Seven SSR markers were chosen based upon their joint ability to discriminate among varieties and, following selection of alternative primers for three of the markers, were combined into a single multiplex with no overlap of allele sizes. A survey of 120 seeds of each of 23 registered Canadian flax varieties indicated that polymorphism within varieties was common and may add a layer of complexity to variety identification and, in particular, to seed purity analysis. Examination of large numbers of individual reference seeds is recommended when constructing databases to ensure that polymorphism that may exist within varieties is sufficiently represented.

Accurate and efficient means to determine the variety composition of wheat composites are required to provide assurances in the grain handling system. We developed a variety identification system for hexaploid wheat (Triticum aestivum L.) that operates with a simple workflow and can sustain a high level of throughput. A set of 32 SNP genotyping assays was developed for use with the TaqMan OpenArray genotyping system. A reference profile database was constructed based on a survey of these markers in 24 kernels of each of 128 wheat varieties representing four market classes. Custom software was written to generate allele profiles from exported OpenArray fluorescence data and query those profiles against the reference database to produce variety calls. We demonstrated the effectiveness of the system in blind analyses of 192 kernels comprising 119 varieties. Each kernel was correctly identified with the exception of two that were called as unidentified and were later confirmed to have uncommon variant profiles not previously encountered.

Perry, D. J., Fernando, U. and Lee, S-J. 2014. Simple sequence repeat-based identification of Canadian malting barley varieties. Can. J. Plant Sci. 94: 485–496. Practical and reliable means to identify barley varieties are required to provide assurances in segregated grain handling and for quality control in the malting and brewing industry. A set of 10 simple sequence repeat (SSR) markers was selected to differentiate among malting barley varieties grown in Canada. Modification of some PCR primers permitted assembly into two five-marker multiplexes that may be examined simultaneously using an electrophoresis-based DNA analyzer. These markers were surveyed in multiple individual kernels of each of 48 barley varieties grown in Canada, including 31 malting varieties and 17 popular feed varieties. Variation within varieties was common and three general categories of intra-variety polymorphism were recognized: (1) primary biotypes, which were characterized by a fairly even distribution of two alleles at one or more marker loci and complete mixture of allele combinations among the polymorphic loci; (2) uncommon, distinctly different variants; and (3) putative recent SSR mutations. Differentiation among varieties was complete with the exception of one pair of related six-row feed varieties (AC Rosser and AC Ranger) that was indistinguishable and one group of three very closely related two-row malting varieties (CDC Kendall, CDC PolarStar and Norman) that, on an individual-kernel basis, were only partially distinguishable using these markers. Simple, rapid individual-kernel DNA preparation methods were also developed for use in conjunction with the multiplexed markers to provide a convenient, effective and relatively inexpensive tool that may be used for barley variety identification, purity analysis or quantification of variety mixtures.

Demeke, T. and Perry, D. J. 2014. Low level presence of unapproved biotech materials: Current status and capability of DNA-based detection methods. Can. J. Plant Sci. 94: 497–507. In agricultural biotechnology, low level presence (LLP) of recombinant DNA plant material is defined as the unintended presence of trace levels of a specific genetically engineered (GE) or biotechnology-derived material which in most instances has been authorized for use as food or feed in at least one country. Asynchronous authorizations of GE products have prompted testing for the GE content in an assortment of agricultural products for the purpose of facilitating international grain trade. Low level presence of some unauthorized GE materials identified in non-GE grains, oilseeds and food stuffs has negatively impacted grain trade. Other factors contributing to a negative impact on grain trade due to LLP of GE material include zero tolerance policies and slow regulatory approval processes for some countries. This element alone heightens the need for accurate, reliable and cost-effective detection methods. As the number of biotech events increases, the challenge of handling LLP of unapproved GE materials poses an even greater challenge. Polymerase chain reaction (PCR) is widely used for detection and quantification of GE events. Accuracy of PCR-based testing of GE events is affected by variation in sampling, sample preparation and various confounders associated with testing methods. Challenges when using PCR detection and quantification methods for the detection of LLP of GE events are the focus of this review as well as background information and recent examples of occurrence and suggestions to mitigate LLP as it relates to GE materials in grain trade.

The global expansion in the development and cultivation of genetically modified (GM) crops has increased international concern about adventitious presence of GM materials in non-GM seeds and grains. GM events in canola, corn, soybean, cotton, flax, papaya, potato, squash, sugar beet, and tomato have received regulatory approval in Canada. However, GM cultivars are only in commercial production for canola, corn and soybean. More than 30 GM events have been approved in these three crops. Cases of unapproved adventitious presence of GM materials that have impacted grain trading and handling in Canada and other countries include StarLink™ corn, GT200 canola, GM canola in mustard and recently Bt10 corn. Some countries have established tolerance and traceability requirements for adventitious presence of GMOs, while others are in the process of developing or adopting legislation. The threshold for labeling of adventitious presence of approved GM material in non-GM grain varies from 0.9% (e.g., EU) to 5% (e.g., Japan). Progress has been made in the development of DNA- and protein-based GMO detection methods. However, only a limited number of these detection methods have been internationally validated. The challenges for detection methods include sampling, a lack of certified reference material, a lack of DNA sequence information for the design of event-specific primers, and the sheer number of individual events that may be present and tested for. Current efforts by ISO and CEN will be valuable for establishing harmonized and standardized GMO detection methods. Key words: List of GM events, cases of AP, tolerance, traceability, detection methods, challenges

Accurate and reliable means of variety identification are necessary to assess purity of seed supplies, to support claims relating to plant breeders' rights and, in Canada, to provide quality assurances in the grain handling system. A single, multiplexed set of seven simple-sequence-repeat (SSR) markers was found to uniquely identify all 18 durum wheat varieties that have been developed in Canada and are currently, or were formerly, registered for commercial production. Significant features of this multiplexed set include an allele that is specific, within Canadian durum varieties, to those having high gluten strength, and redundancy that was included in an effort to increase the capacity to accommodate future varieties. In combination with a reasonably rapid individual-kernel DNA extraction protocol and automated allele calling, this marker system offers a higher resolution alternative to complement established protein-based variety identification methods.

cDNA-based sequence-tagged-site (STS) markers were used to examine the genetic composition of three mature, layer-origin populations of black spruce (Picea mariana (Mill.) BSP), which were the result of logging operations in the first half of the 20th century, and compare them with four mature, seedling-origin populations that regenerated naturally following fire. The amount of STS-marker variation revealed in these populations was very similar to that previously observed in a rangewide panel of black spruce trees. There was little differentiation among populations, and no significant differences in heterozygosities, numbers of alleles, or fixation indices were evident between layer-origin and fire-origin stands. Likewise, when mating-system parameters were estimated in one population of each of these two types, no significant differences were found; outcrossing was essentially complete with no evidence of mating among relatives. The estimated correlation of paternity within progeny arrays was about 17 and 13% in the fire-origin and layer-origin stands, respectively, but again the observed difference was not statistically significant. At least at the current scale of sampling, silvicultural practices that result in stand replacement by layer-origin advance regeneration appear not to have had negative impact upon the genetic diversity or level of inbreeding in second-growth black spruce stands.

Black spruce (Picea mariana [B.S.P.] Mill.) and red spruce (Picea rubens Sarg.) are two conifer species known to hybridize naturally in northeastern North America. We hypothesized that there is a progenitor-derivative relationship between these two taxa and conducted a genetic investigation by using sequence-tagged-site markers of expressed genes. Based on the 26 sequence-tagged-site loci assayed in this study, the unbiased genetic identity between the two taxa was quite high with a Value of 0.920. The mean number of polymorphic loci, the mean number of alleles per polymorphic locus, and the average observed heterozygosity were lower in red spruce (P = 35%. A(P) = 2.1. H-O = 0.069) than in black spruce (P = 54%, A(P) = 2.9, H-O = 0.103). No unique alleles were found in red spruce, and the observed patterns of allele distribution indicated that the genetic diversity of red spruce was essentially a subset of that found in black spruce. When considered in combination with ecological evidence and simulation results, these observations clearly support the existence of a progenitor-derivative relationship and suggest that the reduced level of genetic diversity in red spruce may result from allopatric speciation through glaciation-induced isolation of a preexisting black spruce population during the Pleistocene era. Our observations signal a need for a thorough reexamination of several conifer species complexes in which natural hybridization is known to occur.

We examined the amount and nature of variation revealed by cDNA-based sequence-tagged-site (STS) markers in Norway spruce (Picea abies (L.) Karst.) using 39 pairs of heterologous primers that were based upon arbitrary genes in black spruce (Picea mariana (Mill.) B.S.P.). A panel of 22 diverse Norway spruce genotypes was screened for variation that could be observed directly using standard agarose gel electrophoresis, without additional manipulation of amplification products. Examination of marker segregation among haploid megagametophytes revealed that nine markers behaved in a codominant manner, two markers had codominant length polymorphisms and null alleles, and four others had dominant length polymorphisms. DNA sequencing of codominant alleles at seven loci indicated that most insertions/deletions (indels) were in noncoding regions and that alleles often differed by the presence or absence of direct repeats that ranged in size from three to 23 bp. The nine markers that showed exclusively codominant polymorphisms in Norway spruce had an average observed heterozygosity of 0.30 and an average of 2.9 alleles in the panel of 22 trees. These levels of variation are similar to those previously found for similar sets of markers in other spruces, and appear to be at least as high as those revealed by polymorphic allozyme markers in Norway spruce. Polymorphisms at one STS locus suggested a higher affinity between Norway spruce and white spruce (Picea glauca (Moench) Voss) than between either of these spruces and black spruce. The STS markers described in this report should be useful in a variety of applications in Norway spruce, including population studies and genome mapping.

Sequence-tagged-site primers, previously developed based upon black spruce ( Picea mariana ) cDNA sequences, were tested for their ability to direct specific amplification in two individuals of each of 12 additional conifer species. Nearly all (95–97%) of the primers functioned well in congeneric trials, while a lower proportion (21–33%) scored positively in other Pinaceae genera. Outside of the Pinaceae, amplification of homologous products was not achieved. Products from the various species often differed in size from their homologs in black spruce. In one case a large difference in size was due to the lack of an intron in a jack pine product while in several other cases the differences were due to the presence or absence of large direct repeats in the DNA sequences. Length polymorphism was occasionally evident between the two individuals examined of a given species. We investigated marker polymorphism in detail in a panel of 15 white spruce ( Picea glauca ) trees. Allelic segregation among haploid megagametophytes was revealed directly at 16 loci by standard agarose-gel electrophoresis without any additional manipulation of amplification products. Polymorphisms observed at 12 of these loci were exclusively co-dominant. For this subset of 12 loci, the average number of alleles was 3.2 and the average observed heterozygosity was 0.37.

Sequence-tagged-site (STS) markers of arbitrary genes were investigated in black spruce [Picea mariana (Mill.) B.S.P.]. Thirty-nine pairs of PCR primers were used to screen diverse panels of haploid and diploid DNAs for variation that could be detected by standard agarose gel electrophoresis without further manipulation of amplification products. Codominant length polymorphisms were revealed at 15 loci. Three of these loci also had null amplification alleles as did 3 other loci that had no apparent product-length variation. Dominant length polymorphisms were observed at 2 other loci. Alleles of codominant markers differed in size by as little as 1 bp to as much as an estimated 175 bp with nearly all insertions/deletions found in noncoding regions. Polymorphisms at 3 loci involved large (33 bp to at least 114 bp) direct repeats and similar repeats were found in 7 of 51 cDNAs sequenced. Allelic segregation was in accordance with Mendelian inheritance and linkage was detected for 5 of 63 pairwise combinations of loci tested. Codominant STS markers of 12 loci revealed an average heterozygosity of 0.26 and an average of 2.8 alleles in a range-wide sample of 22 trees.

The alcohol dehydrogenase ( Adh ) gene family is much more complex in Pinus banksiana than in angiosperms, with at least seven expressed genes organized as two tightly linked clusters. Intron number and position are highly conserved between P. banksiana and angiosperms. Unlike angiosperm Adh genes, numerous duplications, as large as 217 bp, were observed within the noncoding regions of P. banksiana Adh genes and may be a common feature of conifer genes. A high frequency of duplication over a wide range of scales may contribute to the large genome size of conifers.

Journal Article SPATIAL GENETIC STRUCTURE IN TWO TAMARACK [LARIX LARICINA (DU ROI) K. KOCH] POPULATIONS WITH DIFFERING ESTABLISHMENT HISTORIES Get access Peggy Knowles, Peggy Knowles School of Forestry and Department of Biology Lakehead University Thunder Bay Ontario P7B 5E1 CANADA Search for other works by this author on: Oxford Academic Google Scholar Daniel J. Perry, Daniel J. Perry Department of Forest Resources University of Minnesota St. Paul MN 55108‐1027 USA Search for other works by this author on: Oxford Academic Google Scholar Heather A. Foster Heather A. Foster School of Forestry and Department of Biology Lakehead University Thunder Bay Ontario P7B 5E1 CANADA Search for other works by this author on: Oxford Academic Google Scholar Evolution, Volume 46, Issue 2, 1 April 1992, Pages 572–576, https://doi.org/10.1111/j.1558-5646.1992.tb02062.x Published: 01 April 1992 Article history Received: 07 August 1990 Accepted: 27 June 1991 Published: 01 April 1992

Pollen pool heterogeneity, which violates an assumption of the mixed-mating model, is a major potential problem in measuring plant mating systems. In this study, isozyme markers were used to examine pollen pool heterogeneity in two natural populations of jack pine, Pinus banksiana Lamb., in northwestern Ontario, Canada. Population multilocus estimates of outcrossing rate ranged from 0.83 to 0.95 and differed significantly between populations. Single-tree multilocus outcrossing rates were found to be homogeneous among trees in both populations. Computer simulation studies indicated that a consanguineous pollen pool (pollen gametes related to the mother tree) was capable of biasing population outcrossing estimates downward. Random pollen pool heterogeneity (uncorrelated with maternal genotypes) did not appear to affect population outcrossing estimates in the simulations. Heterogeneity G-tests and Spearman rank tests showed that pollen pool heterogeneity existed in the two natural populations examined; however, it did not have a major effect on population outcrossing estimates, since the consanguineous pollen pool detected was probably a relatively minor component of the outcross pollen pool in both populations. In addition, heterogeneity G-tests were found to be not sensitive in detecting pollen pool heterogeneity caused by consanguineous pollen pool.

Approximately 100 trees were sampled and mapped in each of three natural Sugar Maple (Acer saccharum Marsh.) stands in northwestern Ontario. Starch-gel electrophoresis was used to resolve five polymorphic enzyme loci from bud tissue and spatial genetic structure was examined in each of the three stands using spatial autocorrelation techniques. A significant small scale genetic structure was detected in all stands and patch widths were inferred to be approximately 20–30 m. A non-random distribution of genotypes may be a reflection of restricted gene flow, selection and/or patchy establishment of genetically distinct cohorts.

Arrays of open-pollinated seeds were assayed for allozyme polymorphisms at four loci (Mdh-1, Me, 6Pg-2, and Pgm) to obtain estimates of outcrossing rates for three eastern white cedar populations in northwestern Ontario, Canada. Multilocus population outcrossing rate estimates were low, ranging from 0.507 to 0.745, with significant heterogeneity among populations. Low stand densities and relatively high selfed embryo survival may have contributed to the low effective outcrossing rates observed. Single-tree multilocus outcrossing rate estimates were obtained for nine trees from one population. When estimated jointly with outcross pollen pool allele frequencies, these outcrossing rate estimates ranged from 0.253 to 1.023 with significant heterogeneity among trees. Key words: mating system, outcrossing rate, Thuja occidentalis L.