Validation of in silico-predicted genic SNPs in white clover (Trifolium repens L.), an outbreeding allopolyploid species

White clover (Trifolium repens L.) is an obligate outbreeding allotetraploid forage legume. Gene-associated SNPs provide the optimum genetic system for improvement of such crop species. An EST resource obtained from multiple cDNA libraries constructed from numerous genotypes of a single cultivar has been used for in silico SNP discovery and validation. A total of 58 from 236 selected sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by genotypic analysis across the parents and progeny of several two-way pseudo-testcross mapping families. The clusters include genes belonging to a broad range of predicted functional categories. Polymorphic SNP-containing ESTs have also been used for comparative genomic analysis by comparison with whole genome data from model legume species, as well as Arabidopsis thaliana. A total of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal locations in Medicago truncatula, which is closely related to white clover within the Trifolieae tribe of the Fabaceae. This analysis provides access to translational data from model species. The efficiency of in silico SNP discovery in white clover is limited by paralogous and homoeologous gene duplication effects, which are resolved unambiguously by the transmission test. This approach will also be applicable to other agronomically important cross-pollinating allopolyploid plant species.

Rice molecular breeding laboratories in the genomics era: Current status and future considerations

Using DNA markers in plant breeding with marker-assisted selection (MAS) could greatly improve the precision and efficiency of selection, leading to the accelerated development of new crop varieties. The numerous examples of MAS in rice have prompted many breeding institutes to establish molecular breeding labs. The last decade has produced an enormous amount of genomics research in rice, including the identification of thousands of QTLs for agronomically important traits, the generation of large amounts of gene expression data, and cloning and characterization of new genes, including the detection of single nucleotide polymorphisms. The pinnacle of genomics research has been the completion and annotation of genome sequences for indica and japonica rice. This information-coupled with the development of new genotyping methodologies and platforms, and the development of bioinformatics databases and software tools-provides even more exciting opportunities for rice molecular breeding in the 21st century. However, the great challenge for molecular breeders is to apply genomics data in actual breeding programs. Here, we review the current status of MAS in rice, current genomics projects and promising new genotyping methodologies, and evaluate the probable impact of genomics research. We also identify critical research areas to "bridge the application gap" between QTL identification and applied breeding that need to be addressed to realize the full potential of MAS, and propose ideas and guidelines for establishing rice molecular breeding labs in the postgenome sequence era to integrate molecular breeding within the context of overall rice breeding and research programs.

Fusion transcript loci share many genomic features with non-fusion loci

Background Fusion transcripts are found in many tissues and have the potential to create novel functional products. Here, we investigate the genomic sequences around fusion junctions to better understand the transcriptional mechanisms mediating fusion transcription/splicing. We analyzed data from prostate (cancer) cells as previous studies have shown extensively that these cells readily undergo fusion transcription. Results We used the FusionMap program to identify high-confidence fusion transcripts from RNAseq data. The RNAseq datasets were from our (N = 8) and other (N = 14) clinical prostate tumors with adjacent non-cancer cells, and from the LNCaP prostate cancer cell line that were mock-, androgen- (DHT), and anti-androgen- (bicalutamide, enzalutamide) treated. In total, 185 fusion transcripts were identified from all RNAseq datasets. The majority (76 %) of these fusion transcripts were ‘read-through chimeras’ derived from adjacent genes in the genome. Characterization of sequences at fusion loci were carried out using a combination of the FusionMap program, custom Perl scripts, and the RNAfold program. Our computational analysis indicated that most fusion junctions (76 %) use the consensus GT-AG intron donor-acceptor splice site, and most fusion transcripts (85 %) maintained the open reading frame. We assessed whether parental genes of fusion transcripts have the potential to form complementary base pairing between parental genes which might bring them into physical proximity. Our computational analysis of sequences flanking fusion junctions at parental loci indicate that these loci have a similar propensity as non-fusion loci to hybridize. The abundance of repetitive sequences at fusion and non-fusion loci was also investigated given that SINE repeats are involved in aberrant gene transcription. We found few instances of repetitive sequences at both fusion and non-fusion junctions. Finally, RT-qPCR was performed on RNA from both clinical prostate tumors and adjacent non-cancer cells (N = 7), and LNCaP cells treated as above to validate the expression of seven fusion transcripts and their respective parental genes. We reveal that fusion transcript expression is similar to the expression of parental genes. Conclusions Fusion transcripts maintain the open reading frame, and likely use the same transcriptional machinery as non-fusion transcripts as they share many genomic features at splice/fusion junctions.

Survival and reproduction of Tribolium castaneum (Herbst), Rhyzopertha dominica (F.) and Sitophilus oryzae (L.) following periods of starvation

This study determined the starvation tolerance of Tribolium castaneum (Herbst), Rhyzopertha dominica (F.) and Sitophilus oryzae (L.) in terms of both adult survival and reproduction, the impact of starvation on reproduction not having been studied before. Experiments were conducted at 30°C and 55% or 70% r.h. using a laboratory strain and a field strain of each species. The number of progeny was a better indicator of the impact of starvation on a species than adult survival. Tribolium castaneum was the most tolerant species, requiring up to 35 d starvation before no progeny were produced. Rhyzopertha dominica and S. oryzae required up to 8 d starvation before no progeny were produced. The results suggest that hygiene will have a greater impact on populations of S. oryzae and R. dominica than T. castaneum.

Acacia nilotica ssp. indica (L.) Willd. ex Del. (Mimosaceae)

Prickly acacia, a Weed of National Significance or WONS, is a serious problem in Queensland particularly the Mitchell grass downs where it was once planted to provide shade for livestock. The chapter summarises current knowledge about the taxonomy, biology, distribution, ecology, impacts and biological control of the weed. Queensland has been trying to achieve biological control of prickly acacia since 1980 when it began foreign exploration in Pakistan. Since then further exploration was undertaken in Kenya, South Africa and presently India. Six insects have been released in Queensland but only two of these are established. Greater emphasis is being placed on climate matching, plant response to herbivory and genotype matching in present work and it is hoped that this approach will allow more rigorous evaluations of agent performance and better understanding of reasons for success or failure of agents.

Detection of equine herpesvirus type 1 using a real-time polymerase chain reaction.

Equid herpesvirus 1 (EHV1) is a major disease of equids worldwide causing considerable losses to the horse industry. A variety of techniques, including PCR have been used to diagnose EHV1. Some of these PCRs were used in combination with other techniques such as restriction enzyme analysis (REA) or hybridisation, making them cumbersome for routine diagnostic testing and increasing the chances of cross-contamination. Furthermore, they involve the use of suspected carcinogens such as ethidium bromide and ultraviolet light. In this paper, we describe a real-time PCR, which uses minor groove-binding probe (MGB) technology for the diagnosis of EHV1. This technique does not require post-PCR manipulations thereby reducing the risk of cross-contamination. Most importantly, the technique is specific; it was able to differentiate EHV1 from the closely related member of the Alphaherpesvirinae, equid herpesvirus 4 (EHV4). It was not reactive with common opportunistic pathogens such as Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Enterobacter agglomerans often involved in abortion. Similarly, it did not react with equine pathogens such as Streptococcus equi, Streptococcus equisimilis, Streptococcus zooepidemicus, Taylorella equigenitalis and Rhodococcus equi, which also cause abortion. The results obtained with this technique agreed with results from published PCR methods. The assay was sensitive enough to detect EHV1 sequences in paraffin-embedded tissues and clinical samples. When compared to virus isolation, the test was more sensitive. This test will be useful for the routine diagnosis of EHV1 based on its specificity, sensitivity, ease of performance and rapidity.

Completion of the genome sequence of Lettuce necrotic yellows virus, type species of the genus Cytorhabdovirus

We completed the genome sequence of Lettuce necrotic yellows virus (LNYV) by determining the nucleotide sequences of the 4a (putative phosphoprotein), 4b, M (matrix protein), G (glycoprotein) and L (polymerase) genes. The genome consists of 12,807 nucleotides and encodes six genes in the order 3′ leader-N-4a(P)-4b-M-G-L-5′ trailer. Sequences were derived from clones of a cDNA library from LNYV genomic RNA and from fragments amplified using reverse transcription-polymerase chain reaction. The 4a protein has a low isoelectric point characteristic for rhabdovirus phosphoproteins. The 4b protein has significant sequence similarities with the movement proteins of capillo- and trichoviruses and may be involved in cell-to-cell movement. The putative G protein sequence contains a predicted 25 amino acids signal peptide and endopeptidase cleavage site, three predicted glycosylation sites and a putative transmembrane domain. The deduced L protein sequence shows similarities with the L proteins of other plant rhabdoviruses and contains polymerase module motifs characteristic for RNA-dependent RNA polymerases of negative-strand RNA viruses. Phylogenetic analysis of this motif among rhabdoviruses placed LNYV in a group with other sequenced cytorhabdoviruses, most closely related to Strawberry crinkle virus.

Limb-loss in pond-reared blue swimmer crabs Portunus pelagicus (L.): Effect on growth in an indoor shedding system

Limb-loss in crustaceans can reduce moult increment and delay or advance the timing of moulting, both aspects that are likely to impact upon soft-shell crab production. Pond-reared blue swimmer crabs Portunus pelagicus were harvested and maintained in a crab shedding system. The wet weight, carapace width (CW) and the occurrence of limb-loss were assessed before stocking in the shedding system and after each of the next three moults. Many of the crabs were initially missing one or two limbs and these did not grow as much as the crabs that were intact at the start of the trial. Despite its strong correlation with wet weight, CW changes proved to be misleading. Limb-loss reduced the %CW increment but not the per cent weight increment (where the later is calculated from the actual pre-moult weight). Pre-moult weight explained much of the variation in post-moult weight, with crabs moulting to approximately double their weight. Limb-loss reduced 'growth' and production from the pond because it reduced pre-moult weight but limb-loss did not alter the weight change on shedding a given weight of crabs, although some of that change now included regeneration of limbs. One can hypothesize that much of the size variation seen in pond-reared crabs may be due to accumulated effects of repeated limb-loss, rather than genetic variation.

Identification of QTL for sugar-related traits in a sweet x grain sorghum (Sorghum bicolor L. Moench) recombinant inbred population

QTL for stem sugar-related and other agronomic traits were identified in a converted sweet (R9188) × grain (R9403463-2-1) sorghum population. QTL analyses were conducted using phenotypic data for 11 traits measured in two field experiments and a genetic map comprising 228 SSR and AFLP markers grouped into 16 linkage groups, of which 11 could be assigned to the 10 sorghum chromosomes (SBI-01 to SBI-10). QTL were identified for all traits and were generally co-located to five locations (SBI-01, SBI-03, SBI-05, SBI-06 and SBI-10). QTL alleles from R9188 were detected for increased sucrose content and sugar content on SBI-01, SBI-05 and SBI-06. R9188 also contributed QTL alleles for increased Brix on SBI-05 and SBI-06, and increased sugar content on SBI-03. QTL alleles from R9403463-2-1 were found for increased sucrose content and sucrose yield on SBI-10, and increased glucose content on SBI-07. QTL alleles for increased height, later flowering and greater total dry matter yield were located on SBI-01 of R9403463-2-1, and SBI-06 of R9188. QTL alleles for increased grain yield from both R9403463-2-1 and R9188 were found on SBI-03. As an increase in stem sugars is an important objective in sweet sorghum breeding, the QTL identified in this study could be further investigated for use in marker-assisted selection of sweet sorghum.

An assessment of the genetic relationship between sweet and grain sorghums, within Sorghum bicolor ssp. bicolor (L.) Moench, using AFLP markers

Compared to grain sorghums, sweet sorghums typically have lower grain yield and thick, tall stalks which accumulate high levels of sugar (sucrose, fructose and glucose). Unlike commercial grain sorghum (S. bicolor ssp. bicolor) cultivars, which are usually F1 hybrids, commercial sweet sorghums were selected as wild accessions or have undergone limited plant breeding. Although all sweet sorghums are classified within S. bicolor ssp. bicolor, their genetic relationship with grain sorghums is yet to be investigated. Ninety-five genotypes, including 31 sweet sorghums and 64 grain sorghums, representing all five races within the subspecies bicolor, were screened with 277 polymorphic amplified fragment length polymorphism (AFLP) markers. Cluster analysis separated older sweet sorghum accessions (collected in mid 1800s) from those developed and released during the early to mid 1900s. These groups were emphasised in a principle component analysis of the results such that sweet sorghum lines were largely distinguished from the others, particularly by a group of markers located on sorghum chromosomes SBI-08 and SBI-10. Other studies have shown that QTL and ESTs for sugar-related traits, as well as for height and anthesis, map to SBI-10. Although the clusters obtained did not group clearly on the basis of racial classification, the sweet sorghum lines often cluster with grain sorghums of similar racial origin thus suggesting that sweet sorghum is of polyphyletic origin within S. bicolor ssp. bicolor.

Identification of small juvenile scombrids from northwest tropical Australia using mitochondrial DNA cytochrome b sequences

Small juveniles of the nine species of scombrids in Australian waters are morphologically similar to one another and, consequently, difficult to identify to species level. We show that the sequence of the mitochondrial DNA cytochrome b gene region is a powerful tool for identification of these young fish. Using this method, we identified 50 juvenile scombrids collected from Exmouth Bay, Western Australia. Six species of scombrids were apparent in this sample of fish: narrow-barred Spanish mackerel (Scomberomorus commerson), Indian mackerel (Rastrelliger kanagurta), frigate tuna (Auxis thazard), bullet tuna (Auxis rochei), leaping bonito (Cybiosarda elegans), and kawakawa (Euthynnus affinis). The presence of Indian mackerel, frigate tuna, leaping bonito, and kawakawa is the first indication that coastal waters may be an important spawning habitat for these species, although offshore spawning may also occur. The occurrence of small juvenile S. commerson was predicted from the known spawning patterns of that species, but other mackerel species (Scomberomorus munroi, Scomberomorus queenslandicus, Scomberomorus semifasiciatus) likely to be spawning during the sampling period were not detected among the 50 small juveniles analyzed here.

Genetic analysis of foot-and-mouth disease virus serotype A of Indian origin and detection of positive selection and recombination in leader protease- and capsid-coding regions

The leader protease (L-pro) and capsid-coding sequences (P1) constitute approximately 3 kb of the foot-and-mouth disease virus (FMDV). We studied the phylogenetic relationship of 46 FMDV serotype A isolates of Indian origin collected during the period 1968-2005 and also eight vaccine strains using the neighbour-joining tree and Bayesian tree methods. The viruses were categorized under three major groups - Asian, Euro-South American and European. The Indian isolates formed a distinct genetic group among the Asian isolates. The Indian isolates were further classified into different genetic subgroups (<5% divergence). Post-1995 isolates were divided into two subgroups while a few isolates which originated in the year 2005 from Andhra Pradesh formed a separate group. These isolates were closely related to the isolates of the 1970s. The FMDV isolates seem to undergo reverse mutation or onvergent evolution wherein sequences identical to the ancestors are present in the isolates in circulation. The eight vaccine strains included in the study were not related to each other and belonged to different genetic groups. Recombination was detected in the L-pro region in one isolate (A IND 20/82) and in the VP1 coding 1D region in another isolate (A RAJ 21/96). Positive selection was identified at aa positions 23 in the L-pro (P<0.05; 0.046*) and at aa 171 in the capsid protein VP1 (P<0.01; 0.003**).

Helix-coil stability constants for amino acids in nonaqueous solvents. Studies of random poly(-benzyl-L-glutamate-co-L-alanine) and poly(-benzyl-L-glutamate-co-L-leucine) in a mixed solvent

The host-guest technique has been applied to the determination of the helix-coil stability constants of two naturally occurring amino acids, L-alanine and L-leucine, in a nonaqueous solvent system. Random copolymers containing L-alanine and L-leucine, respectively, as guest residues and -benzyl-L-glutamate as the host residue were synthesized. The polymers were fractionated and characterized for their amino acid content, molecular weight, and helix-coil transition behavior in a dichloroacetic acid (DCA)-1,2-dichloroethane (DCE) mixture. Two types of helix-coil transitions were carried out on the copolymers: solvent-induced transitions in DCA-DCE mixtures at 25°C and thermally induced transitions in a 82:18 (wt %) DCA-DCE mixture. The thermally induced transitions were analyzed by statistical mechanical methods to determine the Zimm-Bragg parameters, and s, of the guest residues. The experimental data indicate that, in the nonaqueous solvent, the L-alanine residue stabilizes the -helical conformation more than the L-leucine residue does. This is in contrast to their behavior in aqueous solution, where the reverse is true. The implications of this finding for the analysis of helical structures in globular proteins are discussed.

Crystallization kinetics of poly(l-lactic acid)

The morphology and crystal growth of poly(l-lactic acid), PLLA have been studied from the melt as a function of undercooling and molecular weight using hot stage microscopy. Attention has been given to the application of growth rate equation on the growth rate data of PLLA and thus various nucleation parameters have been calculated. The criteria of Regime I and Regime II types of crystallization has been applied for the evaluation of substrate lengths.

J. L. Runebergs samlade skrifter i textkritisk belysning

The Collected Works of J. L. Runeberg from the Viewpoint of Textual Scholarship The theoretical framework of this dissertation builds on textual scholarship. The dissertation explores the history of Runeberg’s publications and his relations with his publishers, from his debut and the first editions, through the editions of collected works published during the course of his life, to the later commercial editions, including the critical edition, published in 1933–2005 by the Svenska Vitterhetssamfundet (The Swedish Society for Belles Lettres) and The Svenska litteratursällskapet i Finland (The Society of Swedish Literature in Finland). The various editions of Runeberg’s collected works are situated in their respective critical traditions, from the 19th century German Ausgabe letzter Hand, to the influence of Anglo-American bibliography on Swedish textual criticism in the late 20th century. By making use of primary material previously not used for research purposes, the author is able to present a new view on Runebergian publishing history, including Runeberg’s fees and his relations with the censor authorities. There are indications that his Finnish publishers could not bear the cost of his sizable fees, that were in proportion neither with the book market in Finland nor with the numbers of copies sold. Apart from a certain body of editions the primary material is comprised of correspondences, publishing contracts, printing house invoices, as well as censor authority records. One of the conclusions drawn is that the early and detailed biography, Biografiska anteckningar om Johan Ludvig Runeberg (Biographical Notes on …) by J. E. Strömborg is not reliable in matters concerning publishing history, and that this work has been used far too uncritically. The history of the critical edition gets a chapter of its own, based on primary material in Swedish and Finnish archives. Finally, the author analyses the critical choices, made primarily in the critical edition, and uses examples from the commercial editions to study the editors’ interventions over time, from the 1850s to the 1920s. The changes to the text are usually small and subtle, but cumulative – and in some cases, crucial for the interpretation of the work. One objective of textual scholarship should be to examine the publishing history of a single work or of an author’s œuvre, and another to pay attention both to changes in a work as such and to the shifts of meaning they might entail.