Gregory W. Peterson

Allelic reduction and genetic shift in the Canadian hard red spring wheat germplasm released from 1845 to 2004

Analysis of genetic diversity changes in existing gene pools of cultivated crops is important for understanding the impact of plant breeding on crop genetic diversity and developing effective indicators for genetic diversity of cultivated plants. The objective of this study was to assess genetic diversity changes in 75 Canadian hard red wheat (Triticum aestivum L.) cultivars released from 1845 to 2004 using 31 simple sequence repeats (SSRs) markers. A total of 267 SSR alleles were detected, and their allelic frequencies ranged from 0.01 to 0.97, with an average of 0.14. Significant allelic reduction was observed at only four SSR loci for the cultivars released from 1970 onwards. However, 51 alleles (about 19%) present in pre-1910 cultivars were undetected in cultivars released after 1990 and were spread over 27 SSR loci. The proportion of SSR variation accounted for by six breeding periods was 12.5%, by four ancestral families, 16.5%, and by eight breeding programs, 8.4%. The average genetic diversity measured by three different band-sharing methods did not change significantly among cultivars released from different breeding periods, breeding programs, and ancestral families. However, genetic shift was obvious in the cultivars released over the six breeding periods, reflecting well the various breeding efforts over years. These results clearly show the allelic reduction and genetic shift in the Canadian hard red spring wheat germplasm released over time. Consequently, more effort needs to be made to broaden the wheat breeding base and conserve wheat germplasm.

Impact of plant breeding on genetic diversity of the Canadian hard red spring wheat germplasm as revealed by EST-derived SSR markers

Genetic diversity changes in wheat germplasm have been studied using different molecular markers, but little is known about the impact of plant breeding on the transcribed segments of the wheat genome. The objective of this study was to assess diversity changes in 75 Canadian hard red wheat cultivars released from 1845 to 2004 using 37 EST-derived microsatellite (eSSR) markers. These markers were derived from at least 19 transcribed sequences with putative functions assigned and sampled 17 wheat chromosomes. A total of 138 eSSR alleles was detected, and their allelic frequencies ranged from 0.01 to 0.99 with an average of 0.41. Allelic counts were significantly reduced at three loci for cultivars released after 1990. Sixteen alleles at 14 loci in pre-1910 cultivars were lost in cultivars released after 1990. The lost alleles had frequencies ranging from 0.03 to 0.17 and averaging 0.07. Partitioning the eSSR variation showed the four ancestral families accounted for 14.7% of the variation, followed by the six breeding periods with 12.8% and the eight breeding programs with 5.8%. A genetic shift was observed in the cultivars released over the six breeding periods, reflecting the various breeding efforts. These results illustrate the impact of the Canadian wheat breeding on the transcriptional segments of the wheat genome. These findings, along with those from genomic SSR markers, suggest the Canadian wheat breeding programs have reduced genetic diversity in the hard red spring wheat.

Genetic diversity within a range of cultivars and landraces of flax (Linum usitatissimum L.) as revealed by RAPDs

Analysis of the extent and distribution of genetic diversity incrop plants is essential for optimizing sampling and breedingstrategies. We used random amplified polymorphic DNA (RAPD)markers to assess genetic diversity and relationships in 22 Canadiancultivars, 29 selected world cultivars and 10 landraces of flax(Linum usitatissimum L.). RAPDvariation was generally low and more variation was detected among,than within, the investigated flax accessions. Based on 53 variableRAPD loci observed for the 61 accessions, the landraces had a lowerproportion of fixed recessive RAPD loci (0.427) (i.e.,more genetic variation) than all of the flax cultivars examined(0.492). The linseed cultivars had a lower proportion ofrecessive loci (0.469) than the fiber flax cultivars(0.529). Canadian linseed cultivars had a lower proportionof recessive loci (0.465) than the selected world flaxcultivars (0.512). A trend was also observed that the rateof loss in genetic variation in Canadian flax breeding programs overthe last fifty years was approximately two variable loci per 100 lociper 10 years. Clustering analyses based on similarity estimatesshowed that the fiber cultivars were more related (or similar toeach other) and were classified as a homogeneous group. All ofthe linseed cultivars were clustered in diverse groups with the ninelandrace accessions. Implications of these findings for flax breedingand germplasm management are discussed.

RAPD analysis of genetic relationships of seven flax species in the genus Linum L

The wild progenitor of the cultivated flax (Linumusitatissimum L.) has been long hypothesized to beL. angustifolium Huds., largely fromseveral phytogeographic cytogenetic and phenotypic studies, but no molecularstudies on the issue are found. In this study, we genotyped 12 flax accessionsrepresenting seven flax species in the genus Linum with 527RAPD loci from 29 informative RAPD primers and analyzed their geneticrelationships with simple matching, Dices and Jaccards similaritycoefficients. Large RAPD variations were found among the flax species.L. usitatissimum andL. angustifolium had a higher RAPDsimilarity than the other pairs of flax species and these two species wereconsistently clustered in the same group with all of the similarity coefficientsused. This molecular finding provides an additional support for the hypothesisof L. angustifolium as the wildprogenitor of cultivated flax.

Patterns of AFLP variation in a core subset of cultivated hexaploid oat germplasm

Many core collections have been developed from large collections of crop germplasm, but most of these have not been characterized, particularly using molecular techniques, for germplasm management and utilization. We have attempted to characterize a structured sample representing a world collection of 11,622 cultivated hexaploid oat accessions in the hope of understanding the genetic structure of the world collection. The amplified fragment length polymorphism (AFLP) technique was applied to screen 670 accessions representing 79 countries and one group of uncertain origin. For each accession, 170 AFLP polymorphic bands detected by five AFLP primer pairs were scored. Analyses of the AFLP data showed the effectiveness of the stratified sampling applied in capturing country-wise AFLP variation. The frequencies of polymorphic bands ranged from 0.11 to 0.99, with an average of 0.72. The majority (89.9%) of the AFLP variation resided within accessions of each country, and only 6.2% of the AFLP differences existed among accessions of major geographic regions. Accessions from the Mediterranean region were the most distinct, while those from Russia and the USA were the most diverse. The two distinct groups that were observed were separated largely on the basis of common oat and red oat. Red oat was genetically more diverse than its common and hull-less counterparts, and hull-less oat was more related to common oat than red oat. Landrace and non-landrace accessions displayed similar AFLP variation patterns. These patterns are significant for understanding the domestication of cultivated oat and are useful in classifying the intraspecific diversity of oat germplasm, developing specific core subsets of the oat collection, and exploring new sources of genes for oat improvement.

Genetic Diversity of Canadian and Exotic Potato Germplasm Revealed by Simple Sequence Repeat Markers

Canadian potato germplasm (Solanum tuberosum L.) is unique in its geographic and climatic ranges of adaptation, but little is known about the genetic diversity of the improved Solanum gene pool established over the past century. Simple sequence repeat (SSR) markers were applied to assess the genetic diversity of 114 Canadian and 55 exotic potato accessions. Thirty-six SSR primer pairs were applied and 232 polymorphic bands were scored for each accession. The frequencies of polymorphic bands ranged from 0.01 to 0.98 and averaged 0.35. The proportion of total SSR variation occurring between Canadian and exotic germplasm was 0.6%; among the Canadian cultivars of four major breeding periods 2.7%; among heirloom varieties, modern cultivars and elite breeding lines 4%; and between tetraploid and diploid lines 3.7%. Slightly more diversity was found for exotic, than the Canadian, germplasm. The modern cultivars displayed slightly more diversity than the heirloom varieties and the early cultivars revealed slightly more variation than the recent ones. Clustering 169 accessions revealed more than ten groups, but the groups were not distantly separated. Both the genetically most distinct accessions and the possible genetically duplicated accessions were identified. These findings not only demonstrate the narrow genetic base of the Canadian potato germplasm, but also are useful for managing the existing potato collection and for selecting genetically distinct potato materials to widen the genetic background of the potato gene pool.ResumenEl germoplasma de de papa (Solanum tuberosum L.) canadiense es único en su adaptación geográfica y a variedad de climas, pero se conoce poco acerca de la diversidad genética del acervo genético mejorado de Solanum establecido durante el siglo pasado. Para evaluar la diversidad de 114 accesiones accesiones de papa canadiense y 55 foráneas se utilizaron marcadores de secuencia simple repetida (SSR). Se aplicaron 36 pares de iniciadores SSR y se evaluaron 232 bandas polimórficas para cada accesión. Las frecuencias de bandas polimórficas tuvieron un rango de 0.01 a 0.98 con un promedio de 0.35. La proporción de la variación total de SSR que ocurrió entre el germoplasma canadiense y foráneo fue de 0.6%; entre los cultivares canadienses de los cuatro principales periodos de mejoramiento 2.7%; entre las variedades ancestrales, los cultivares modernos, las líneas élite de mejoramiento 4%; y entre las líneas tetraploides y diploides 3.7%. Se ha encontrado una diversidad ligeramente mayor para el germoplasma foráneo que para el canadiense. Los cultivares modernos mostraron una diversidad ligeramente mayor que las variedades ancestrales y los cultivares antiguos revelaron una ligera mayor variación que los recientes. La agrupación de 169 accesiones reveló más de 10 grupos, pero éstos no estaban separados distantemente. Tanto las accesiones genéticamente más distintas como las posiblemente duplicadas fueron identificadas. Estos hallazgos no sólo demostraron la estrecha base genética del germoplasma canadiense de papa, sino que también son útiles para administrar la colección existente de papa, y para seleccionar materiales genéticamente distintos y ampliar la base genética del acervo genético de papa.

Developing genomic resources in two Linum species via 454 pyrosequencing and genomic reduction

Recent advances in next‐generation DNA sequencing (NGS) have enhanced the development of genomic resources such as contigs or single‐nucleotide polymorphisms (SNPs) for evolutionary studies of a nonmodel species with a complex and unsequenced genome. This study presents an application of a NGS technique in combination with genomic reduction and advanced bioinformatics tools to identify contigs and SNPs from multiple samples of two Linum species. A full Roche 454 GS FLX run of 16 diverse Linum samples representing cultivated flax (Linum usitatissimum L.) and its wild progenitor (Linum bienne Mill.) generated approximately 1.6 million sequence reads with a total length of 498 Mbp. Application of the computational pipeline de novo identification of alleles identified 713 contigs and 1067 SNPs. A blast search revealed alignments of all 713 contigs with 491 existing Linum scaffolds and gene annotations associated with 512 contigs. Sanger sequencing confirmed 95% of 79 selected contigs and 94% of 272 SNPs and identified 211 new SNPs and 19 new indels. The scored 454 SNP data were highly imbalanced for assayed samples. These findings not only are useful for evolutionary studies of Linum species but also help to illustrate the utility of NGS technologies in SNP discovery for nonmodel organisms.

Increasing Genome Sampling and Improving SNP Genotyping for Genotyping-by-Sequencing with New Combinations of Restriction Enzymes

Genotyping-by-sequencing (GBS) has emerged as a useful genomic approach for exploring genome-wide genetic variation. However, GBS commonly samples a genome unevenly and can generate a substantial amount of missing data. These technical features would limit the power of various GBS-based genetic and genomic analyses. Here we present software called IgCoverage for in silico evaluation of genomic coverage through GBS with an individual or pair of restriction enzymes on one sequenced genome, and report a new set of 21 restriction enzyme combinations that can be applied to enhance GBS applications. These enzyme combinations were developed through an application of IgCoverage on 22 plant, animal, and fungus species with sequenced genomes, and some of them were empirically evaluated with different runs of Illumina MiSeq sequencing in 12 plant species. The in silico analysis of 22 organisms revealed up to eight times more genome coverage for the new combinations consisted of pairing four- or five-cutter restriction enzymes than the commonly used enzyme combination PstI + MspI. The empirical evaluation of the new enzyme combination (HinfI + HpyCH4IV) in 12 plant species showed 1.7–6 times more genome coverage than PstI + MspI, and 2.3 times more genome coverage in dicots than monocots. Also, the SNP genotyping in 12 Arabidopsis and 12 rice plants revealed that HinfI + HpyCH4IV generated 7 and 1.3 times more SNPs (with 0–16.7% missing observations) than PstI + MspI, respectively. These findings demonstrate that these novel enzyme combinations can be utilized to increase genome sampling and improve SNP genotyping in various GBS applications.

Assessment of bulking strategies for RAPD analyses of flax germplasm

Effectiveness of several bulking strategies was empirically assessed in detecting RAPD variations and determining genetic relationships of five flax (Linum usitatissimum L.) landrace accessions. Bulking ten individuals before and after DNA isolations generated consistent RAPD variations. About 30% of the polymorphic RAPD loci observed in the plant-by-plant (PBP) sample were difficult to score and/or undetected in the bulked samples of the same accession. Heterogeneity among the six bulked samples of the same accession was observed at 5.6% of the loci scored. The frequency of a specific RAPD band present in those individuals used to form a bulk was at least 1/11 for its detection in the bulked sample. In spite of these limitations, bulking still generated compatible genetic relationships of the five accessions from its PBP sampling.

Biological and genetic characterisation of Phoma macrostoma isolates with bioherbicidal activity

Abstract The fungus Phoma macrostoma Mont. isolate 94-44B was registered as a bioherbicide for control of broadleaved weeds in Canada and the USA in 2011 and 2012, respectively. To obtain the registrations, the fungus had to be characterised both biologically and genetically. The objectives of this study were to demonstrate that bioherbicidal activity was associated with specific genetic markers and to determine whether bioherbicidal activity was a general trait of the species or only selected isolates. A collection of 64 isolates of P. macrostoma was established. A greenhouse bioassay and bioherbicidal-specific primers were used to determine bioherbicidal activity of all isolates. Only isolates originating from Canada thistle demonstrated the ability to reduce dandelion seedlings and display the 853 bp amplicon for the bioherbicidal-specific primer. Bioherbicidal isolates were consistently differentiated from all other isolates with two main genotypic groupings (I and II) arising from internal transcribed spacer (ITS) and amplified fragment length polymorphisms (AFLP) sequence analyses. Using AFLP, two biotypes of bioherbicidal isolates were also differentiated by the presence or absence of an AFLP marker at a single polymorphic locus. The genetic divergence among the bioherbicidal and nonbioherbicidal isolates of P. macrostoma was only 2.21% which was lower than that reported for other related Phoma sp. Other than the bioherbicidal trait, there was no apparent affiliation of the genetics with known varietal types, host or geographic origin. ITS sequence analysis and AFLP fingerprinting may be used as tools to detect bioherbicidal isolates of P. macrostoma.