Yong-Bi Fu

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.

Phylogenetic network of Linum species as revealed by non-coding chloroplast DNA sequences

Crop wild relatives are invaluable sources of novel genes for crop improvement and adaptation to changing environments. We assessed phylogenetic relationships among 29 Linum accessions representing 16 species, including cultivated flax and its progenitor pale flax, based on four non-coding regions of chloroplast DNA sequences. We obtained a cpDNA network showing that these 16 Linum species are appropriately connected based on previously defined taxonomic sections; these connections reflect the same evolutionary pathways as determined from earlier morphological and cytological data. These relationships also support an earlier hypothesis that cultivated flax is probably descended from a single domestication of pale flax plants, apparently for oil usage. The detailed species network not only is significant for understanding evolutionary relationships of Linum species, but also useful for classifying exotic gene pools of cultivated flax as a part of the ongoing exploration of new genetic diversity for flax improvement.

AFLP variation in 25 Avena species

Current molecular characterization of ex situ plant germplasm has placed more emphasis on cultivated gene pools and less on exotic gene pools representing wild relative species. This study attempted to characterize a selected set of germplasm accessions representing various Avena species with the hope to establish a reference set of exotic oat germplasm for oat breeding and research. The amplified fragment length polymorphism (AFLP) technique was applied to screen 163 accessions of 25 Avena species with diverse geographic origins. For each accession, 413 AFLP polymorphic bands detected by five AFLP primer pairs were scored. The frequencies of polymorphic bands ranged from 0.006 to 0.994 and averaged 0.468. Analysis of molecular variance revealed 59.5% of the total AFLP variation resided among 25 oat species, 45.9% among six assessed sections of the genus, 36.1% among three existing ploidy levels, and 50.8% among eight defined genome types. All the species were clustered together according to their ploidy levels. The C genome diploids appeared to be the most distinct, followed by the Ac genome diploid A. canariensis. The Ac genome seemed to be the oldest in all the A genomes, followed by the As, Al and Ad genomes. The AC genome tetraploids were more related to the ACD genome hexaploids than the AB genome tetraploids. Analysis of AFLP similarity suggested that the AC genome tetraploid A. maroccana was likely derived from the Cp genome diploid A. eriantha and the As genome diploid A. wiestii, and might be the progenitor of the ACD genome hexaploids. These AFLP patterns are significant for our understanding of the evolutionary pathways of Avena species and genomes, for establishing reference sets of exotic oat germplasm, and for exploring new exotic sources of genes for oat improvement.

Phenotypic and Molecular (RAPD) Differentiation of Four Infraspecific Groups of Cultivated Flax ( Linum usitatissimum L. subsp. usitatissimum )

Based on agro-botanical characterization data, 3101 accessions of cultivated flax (Linum usitatissimum L. subsp. usitatissimum) from the flax collection held by Plant Gene Resources of Canada (PGRC) were grouped into four infraspecific groups according to the classification proposed by Kulpa and Danert (1962) . The objective of this study was to investigate phenotypic and RAPD variation within and among the four groups to better understand phenotypic and genotypic differentiation within the genepool of cultivated flax. The results of the phenotypic characterization of characters defining the convarieties (capsule dehiscence, plant height, technical stem length and 1000 seed weight) and of other quantitative (petal width, oil content in seeds) and qualitative (RAPD, petal colour, anther colour, petal longitudinal folding and margin folding, ciliation of capsule septa, seed colour) are presented using descriptive statistics. The most frequent convariety in the PGRC genebank was intermediate flax (convar. usitatissimum; 80.7 %), followed by fibre flax (convar. elongatum Vav. et Ell. in Wulff; 13.4%), large-seeded flax (convar. mediterraneum [Vav. ex Ell.] Kulpa et Danert; 5.6%) and dehiscent flax (convar. crepitans [Boenningh.] Kulpa et Danert; 0.3%). Analyses of RAPD data and two qualitative characters (longitudinal and marginal folding of petals) did not show marked differences among the proposed convarieties. However, differences among the convarieties in quantitative traits defining them (plant height, technical stem length and seed size) were considerable. Patterns of variation among the convarieties for other quantitative characters (petal width and seed oil content), as well as the frequencies of character expressions of four qualitative characters (petal colour, anther colour, ciliation of capsule septa and seed colour) were significantly associated with the four proposed convarieties, underlining the phenotypic and genotypic validity of this grouping. The patterns of geographic distribution of the convarieties and important characters showed that certain convarieties dominate in some areas of origin. The infraspecific classification and the presented characterization data increase the transparency of genetic diversity available in cultivated flax and in particular in the PGRC flax collection.

Genetic Diversity Analysis with 454 Pyrosequencing and Genomic Reduction Confirmed the Eastern and Western Division in the Cultivated Barley Gene Pool

Next‐generation DNA sequencing (NGS) technologies can survey sequence variation on a genome‐wide scale, but their utility for crop genetic diversity analysis is poorly known. Many challenges remain in their applications, including sampling complex genomes, identifying single nucleotide polymorphisms (SNPs), and analyzing missing data. This study presented a practical application of the Roche 454 GS FLX Titanium technology in combination with genomic reduction and an advanced bioinformatics tool to analyze the genetic relationships of 16 diverse barley (Hordeum vulgare L.) landraces. A full 454 run generated roughly 1.7 million sequence reads with a total length of 612 Mbp. Application of the computational pipeline called DIAL (de novo identification of alleles) identified 2578 contigs and 3980 SNPs. Sanger sequencing of four barley samples confirmed 85 of the 100 selected contigs and 288 of the 620 putative SNPs and identified 735 new SNPs and 39 new indels. Several diversity analyses revealed the eastern and western division in the barley samples. The division is compatible with those inferred with 156 microsatellite alleles of the same 16 samples and consistent with our current knowledge about cultivated barley. These results help to illustrate the utility of NGS technologies for crop diversity studies. The NGS application also provides a new informative set of genomic resources for barley research.

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.