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Phalaris aquatica L. (phalaris) is a cool-season perennial grass originating from the Mediterranean Basin, north-west Africa and Middle Eastern regions that is used for livestock agriculture mainly in temperate areas with dry summers. It has been the subject of breeding programs in Australia, South America, New Zealand and the USA. Increased knowledge of relationships between wild and cultivated germplasm through use of molecular markers has the potential to facilitate future breeding gains. For this purpose, we conducted an analysis of P. aquatica by using 3905 polymorphic DArTseq SNP markers. Genetic diversity as measured by expected heterozygosity was similar for wild (HE = 0.14; n = 57) and cultivated (HE = 0.13; n = 37) accessions. Diversity in wild germplasm was generally continuous in nature, largely related to geographical location, with a division at the broadest scale into eastern and western clades, with more admixture in the western than the eastern clade. Structure analysis of wild germplasm indicated six subpopulations consistent with country of origin, with some admixture among subpopulations likely resulting from natural and human influences. There were nine subpopulations among wild and cultivated accessions combined. This population structure should be considered if genomic selection is applied in P. aquatica. Analysis of molecular variance indicated that 71% of the genetic variation occurred within subpopulations and 29% among subpopulations. Genetic distances were low among cultivated germplasm from most countries except the USA, which was more distinct. Evaluation of material from the US pool by breeding programs in other countries, and additional material from the less utilised eastern clade, may be worthwhile.

In cases where invasive species are presumed to be strictly exotic, the discovery that the species is also native can be disconcerting for researchers and land managers responsible for eradicating an exotic invasive. Such is the case with reed canarygrass (Phalaris arundinacea), for which decades of misinformation led to the call for nationwide control of this species in the United States. However, native populations were first reported by LaVoie and then later confirmed by Casler with molecular analyses. This, coupled with the discovery by Anderson that this species has been used in weavings by Native Americans for centuries, also made the native forms of interest for protection. Identifying the native status of historic, herbarium specimens via molecular analyses is of great interest to determine localities of native populations for confirmation with extant specimens. Genetic-based methods describing DNA polymorphism of reed canary grass are not well developed. The goal of the presented research is to assess the utility of genomic DNA obtained from historic (herbaria) and extant (fresh) tissue of reed canarygrass and the application of using Diversity Arrays Technology sequencing low density for genetic population studies. [ABSTRACT FROM AUTHOR]

Oil palm (Elaeis guineensisJacq.) is an outbreeding perennial tree crop with long breeding cycles, typically 12 years. Molecular marker technologies can greatly improve the breeding efficiency of oil palm. This study reports the first use of the DArTseq platform to genotype two closely related self-pollinated oil palm populations, namely AA0768 and AA0769 with 48 and 58 progeny respectively. Genetic maps were constructed using the DArT and SNP markers generated in combination with anchor SSR markers. Both maps consisted of 16 major independent linkage groups (2n= 2×= 32) with 1399 and 1466 mapped markers for the AA0768 and AA0769 populations, respectively, including the morphological trait “shell-thickness” (Sh). The map lengths were 1873.7 and 1720.6 cM with an average marker density of 1.34 and 1.17 cM, respectively. The integrated map was 1803.1 cM long with 2066 mapped markers and average marker density of 0.87 cM. A total of 82% of the DArTseq marker sequence tags identified a single site in the published genome sequence, suggesting preferential targeting of gene-rich regions by DArTseq markers. Map integration of higher density focused around the Shregion identified closely linked markers to the Sh, with D.15322 marker 0.24 cM away from the morphological trait and 5071 bp from the transcriptional start of the published SHELLgene. Identification of the Shmarker demonstrates the robustness of using the DArTseq platform to generate high density genetic maps of oil palm with good genome coverage. Both genetic maps and integrated maps will be useful for quantitative trait loci analysis of important yield traits as well as potentially assisting the anchoring of genetic maps to genomic sequences.

High yield is a major objective in canola-breeding programs. We analysed the genetic determinants controlling variation in grain yield in a doubled-haploid (DH) breeding population derived from a single BC1F1 plant from the cross Skipton/Ag-Spectrum//Skipton (designated as the SAgS population). DH lines were evaluated for flowering time and yield in two replicated trials and exhibited significant genetic variation for both traits. Yield showed negative correlation with flowering time; lines that flowered earlier had higher yield than late-flowering lines. A genetic linkage map comprising 7716 DArTseq markers was constructed for the SAgS population, and a ‘bin’ map based on 508 discrete single-position (non-co-segregating) marker loci was used for quantitative trait locus (QTL) analysis. We identified 20 QTLs (LOD ≥2) associated with variation in flowering time and grain yield. Two QTLs (Qy.wwai-A7/Qdtf.wwai-A7/Qfs.wwai-A7 and Qy.wwai-C3a/Qfs.wwai-C3a) appeared repeatedly across experiments, accounting for 4.9–19% of the genotypic variation in flowering time and yield and were located on chromosomes A07 and C03. We identified 22 putative candidate genes for flowering time as well as grain yield, and all were located in a range of 935bp to 2.97 Mb from markers underlying QTLs. This research provides useful information to be used for breeding high-yielding canola varieties by combining favourable alleles for early flowering and higher grain yield at loci on chromosomes A07, C03 and possibly on A06.

Abstract Diversity Arrays Technology (DArT) is a microarray-based DNA marker technique for genome-wide discovery and genotyping of genetic variation. DArT allows simultaneous scoring of hundreds of restriction site based polymorphisms between genotypes and does not require DNA sequence information or site-specific oligonucleotides. This paper demonstrates the potential of DArT for genetic mapping by validating the quality and molecular basis of the markers, using the model plant Arabidopsis thaliana. Restriction fragments from a genomic representation of the ecotype Landsberg erecta (Ler) were amplified by PCR, individualized by cloning and spotted onto glass slides. The arrays were then hybridized with labeled genomic representations of the ecotypes Columbia (Col) and Ler and of individuals from an F2 population obtained from a Col × Ler cross. The scoring of markers with specialized software was highly reproducible and 107 markers could unambiguously be ordered on a genetic linkage map. The marker order on the genetic linkage map coincided with the order on the DNA sequence map. Sequencing of the Ler markers and alignment with the available Col genome sequence confirmed that the polymorphism in DArT markers is largely a result of restriction site polymorphisms.

We present a novel approach to the efficient cloning of telomere-associated sequences and demonstrate its application to the cloning and mapping of these sequences from barley. The method is a modification of the Vectorette PCR technique and allows specific amplification and cloning of subtelomeric sequences. Telomere-associated sequences isolated from barley include hypervariable, repetitive sequences. Polymorphisms detected by subtelomeric markers behaved as Mendelian factors and were mapped to the most distal positions of barley chromosomes 1, 2, and 4.

Anther culture and Hordeum bulbosum-derived doubled haploid (DH) lines of barley (Hordeum vulgare L.) were analyzed for RFLP and RAPD polymorphisms. Polymorphisms were not detected in the anther culture-or H. bulbosum-derived DH lines among 273 RFLP and 89 polymerase chain reaction (PCR)-amplified DNA fragments assayed. It was calculated that base substitution or small deletion/insertion mutations had not been induced among 401 640 by screened. Large deletion/insertion mutations were not observed among 33 Mb screened. Polymorphisms were observed when DNA was digested with the methylation-sensitive restriction enzymes HpaII and MspI: these RFLPs originated primarily from the anther culture-derived doubled haploids. The data indicate that heritable DNA methylation changes had occurred during DH production, particularly with the anther culture method.

Suspension culture cells of barley (Hordeum vulgare L. cv. Klages) were collectec on filters and bombarded with gold particles carrying the Basta® resistance (bar) gene, or both bar and the ß-glucuronidase (uidA) genes in a Biolistic® Particle Accelerator. Filters carrying the bombarded tissues were selected on basal medium with increasing concentrations (maximum of 200 mg I-1) of phosphinothricin. Surviving calli were assayed for the presence of phosphinothricin acetyltransferase activity. All positive calli were shown to have the bar gene integrated into their genome. Nine independent stable transformation events have been analyzed with substantial variation in the integration pattern and transgene copy number. Some albino plantlets were produced, but no mature plants were obtained.

Male (anther culture) and female (Hordeum bulbosum) derived, doubled haploid populations were used to map the barley genome and thus determine the different recombination rates occurring during meiosis in the F1 hybrid donor plants. The anther culture-derived (male recombination) population showed an 18% overall increase in recombination rate. This increased recombination rate was observed for every chromosome and most of the chromosome arms. Examination of linkage distances between individual markers revealed eight segments with significantly higher recombination in the anther culture-derived population, and one in the Hordeum bulbosum-derived population. Very strong distortions of single locus segregations were observed in the anther culture-derived population, but map distances were not affected significantly by these distortions. There were 1.047 and 0.912 recombinations per chromosome in the anther culture and Hordeum bulbosum-derived doubled haploid populations, respectively.

Eukaryotic chromosomes terminate with specialized structures called telomeres. Maintenance of chromosomal ends in most eukaryotes studied to date requires a specialized enzyme, telomerase. Telomerase has been shown to be developmentally regulated in man and a few other multicellular organisms, while it is constitutively expressed in unicellular eukaryotes. Recently, we demonstrated telomerase activity in plant extracts using the PCR-based TRAP (Telomeric Repeat Amplification Protocol) assay developed for human cells. Here we report telomerase activities in two grass species, barley and maize, using a modified, semi-quantitative TRAP assay. Telomerase was highly active in very young immature embryos and gradually declined during embryo development. The endosperm telomerase activity was detectable, but significantly lower than in the embryo and declined during kernel development with no detectable activity in later stages. Telomerase activity in dissected maize embryo axis was several orders of magnitude higher than in the scutellum. Telomerase activity was not detected in a range of differentiated tissues including those with active meristems such as root tips as well as the internode and leaf base. The role of telomerase repression during differentiation and the relationship between chromosome healing and telomerase activity is discussed.

In order to facilitate the map-based cloning of the barley stem rust resistance gene Rpg1, we have demonstrated a high degree of synteny at a micro level between the telomeric regions of barley chromosome 1P and rice chromosome 6. We have also developed and applied a simple and efficient method for selecting useful probes from large Insert genomic YAC and cosmld clones. The gene order within the most terminal 6.5 cM of barley chromosome 1P was compared wtth the most terminal 2.7 cM of rice chromosome 6. Nine rice probes, previously mapped In rice or Isolated from YAC or cosmld clones from this region, were mapped In barley. All, except one, were In synteny with the rice gene order. The exception, probe Y617R, was duplicated in barley. One copy was located on a different chromosome and the other in a non-syntenic position on barley chromosome 1 P. The barley probes from this region could not be mapped to rice, but two of them were inferred to be In a syntenlc location based on their position on a rice YAC. This work demonstrates the utility of applying the results of genetic and physical mapping of the small genome cereal rice to map-based cloning of interesting genes from large genome relatives.