Whole Genome Profiling provides a robust framework for physical mapping and sequencing in the highly complex and repetitive wheat genome

<p>Abstract</p> <p>Background</p> <p>Sequencing projects using a clone-by-clone approach require the availability of a robust physical map. The SNaPshot technology, based on pair-wise comparisons of restriction fragments sizes, has been used recently to build the first physical map of a wheat chromosome and to complete the maize physical map. However, restriction fragments sizes shared randomly between two non-overlapping BACs often lead to chimerical contigs and mis-assembled BACs in such large and repetitive genomes. Whole Genome Profiling (WGP™) was developed recently as a new sequence-based physical mapping technology and has the potential to limit this problem.</p> <p>Results</p> <p>A subset of the wheat 3B chromosome BAC library covering 230 Mb was used to establish a WGP physical map and to compare it to a map obtained with the SNaPshot technology. We first adapted the WGP-based assembly methodology to cope with the complexity of the wheat genome. Then, the results showed that the WGP map covers the same length than the SNaPshot map but with 30% less contigs and, more importantly with 3.5 times less mis-assembled BACs. Finally, we evaluated the benefit of integrating WGP tags in different sequence assemblies obtained after Roche/454 sequencing of BAC pools. We showed that while WGP tag integration improves assemblies performed with unpaired reads and with paired-end reads at low coverage, it does not significantly improve sequence assemblies performed at high coverage (25x) with paired-end reads.</p> <p>Conclusions</p> <p>Our results demonstrate that, with a suitable assembly methodology, WGP builds more robust physical maps than the SNaPshot technology in wheat and that WGP can be adapted to any genome. Moreover, WGP tag integration in sequence assemblies improves low quality assembly. However, to achieve a high quality draft sequence assembly, a sequencing depth of 25x paired-end reads is required, at which point WGP tag integration does not provide additional scaffolding value. Finally, we suggest that WGP tags can support the efficient sequencing of BAC pools by enabling reliable assignment of sequence scaffolds to their BAC of origin, a feature that is of great interest when using BAC pooling strategies to reduce the cost of sequencing large genomes.</p

An affinity-based scoring scheme for predicting DNA-binding activities of modularly assembled zinc-finger proteins

Zinc-finger proteins (ZFPs) have long been recognized for their potential to manipulate genetic information because they can be engineered to bind novel DNA targets. Individual zinc-finger domains (ZFDs) bind specific DNA triplet sequences; their apparent modularity has led some groups to propose methods that allow virtually any desired DNA motif to be targeted in vitro. In practice, however, ZFPs engineered using this ‘modular assembly’ approach do not always function well in vivo. Here we report a modular assembly scoring strategy that both identifies combinations of modules least likely to function efficiently in vivo and provides accurate estimates of their relative binding affinities in vitro. Predicted binding affinities for 53 ‘three-finger’ ZFPs, computed based on energy contributions of the constituent modules, were highly correlated (r = 0.80) with activity levels measured in bacterial two-hybrid assays. Moreover, Kd values for seven modularly assembled ZFPs and their intended targets, measured using fluorescence anisotropy, were also highly correlated with predictions (r = 0.91). We propose that success rates for ZFP modular assembly can be significantly improved by exploiting the score-based strategy described here

An improved predictive recognition model for Cys2-His2 zinc finger proteins

Cys2-His2 zinc finger proteins (ZFPs) are the largest family of transcription factors in higher metazoans. They also represent the most diverse family with regards to the composition of their recognition sequences. Although there are a number of ZFPs with characterized DNA-binding preferences, the specificity of the vast majority of ZFPs is unknown and cannot be directly inferred by homology due to the diversity of recognition residues present within individual fingers. Given the large number of unique zinc fingers and assemblies present across eukaryotes, a comprehensive predictive recognition model that could accurately estimate the DNA-binding specificity of any ZFP based on its amino acid sequence would have great utility. Toward this goal, we have used the DNA-binding specificities of 678 two-finger modules from both natural and artificial sources to construct a random forest-based predictive model for ZFP recognition. We find that our recognition model outperforms previously described determinant-based recognition models for ZFPs, and can successfully estimate the specificity of naturally occurring ZFPs with previously defined specificities

Evaluation of one-shot vaccination protocol for supressing reproductive functions in rams using encapsulated ovalbumin-LHRH-7 protein

The objective of study were to determine the effectiveness of Ovalbumin-LHRH-7 (OL) protein administered with cytosine guanine (CpG) adjuvant and Incomplete Freund’s Adjuvant (IFA), and used one-shot immunization (single-dose vaccination) protocol in which booster dose included in microspheres in rams. Fifty ram lambs at about a year old were used. Treatment groups receiving Ovalbumin LHRH and control contained 10 animals. They were stratified according to age (weeks), live weight and scrotal circumference size, and were randomly assigned to five groups. Scrotal circumference, sexual activities and the numbers of rams having sperm in the ejaculate were affected from treatment (P0.05). Findings clearly demonstrate that the effects of OL immunization on reproductive traits in yearling rams were prominent when it was administered at higher dose and classical one primary and one booster immunization as free protein form. Also we observed that the effect of higher and single dose of OL protein in encapsulated form on reproductive traits had the partial suppressing. CpG adjuvant along with IFA was proved to be an effective adjuvant and could be suggested to be used and alternative to FCA in hormone immunization

The effectiveness of recombinant Ol fusion protein (Ovalbumin-LHRH-7) in supressing reproductive functions when injected in single-dose vaccination protocols with different adjuvants

The objective of this study was to evaluate the effectiveness of recombinant LHRH fusion protein,Ovalbumin-LHRH-7(OL),using a single-dose vaccination protocol in combination with different adjuvants in suppressing reproductive functions in buck kids.For this pur-pose, either a mixture of free OL antigen and encapsulated OL antigen, or encapsulated OL antigen was used. Thirty-nine native buck kids at 12 weeks of age were divided into control (n=7) and treatment groups (n=8 bucks/group).The four treatment groups were formed according to the different vaccine formulations: Group Cp Greceived0.5mgfree OL protein to get her with 1. 0 mg of encapsulated protein with CpG adjuvant. Group mFCA received 0.5mg free OL protein together with 1.0 mg of encapsulated protein with modified Fre-und’s complete adjuvant. Group IS received 1.5 mg encapsulated OL protein with amix of inulin and saponin adjuvants. Group Ism FCA received 1.5mg encapsulated OL protein withamixofinulin, saponin and modified Freund’scomplete adjuvants. Scrotal circumference in CpG and mFCA groups were significantly smaller than that of Control, IS and ISm FCA groups (P<0.05). Numbers and percentage of bucks having spermatozoa in their ejaculate were significantly lowerin Cp G and mFCA groups (P<0.05). OL immunization completely suppressed sperm production, exceptone buck,in CpG and FCA groups (P<0.05).These results imply that it is possible to use OL protein in a single injection protocol for the purpose of immunocastration. Further investigation with a larger number of animals should be carried out to determine the longevity of response to a single injection

A sunflower BAC library suitable for PCR screening and physical mapping of targeted genomic regions

International audienceA sunflower BAC library consisting of 147,456 clones with an average size of 118 kb has been constructed and characterized. It represents approximately 5x sunflower haploid genome equivalents. The BAC library has been arranged in pools and superpools of DNA allowing screening with various PCR-based markers. Each of the 32 superpools contains 4,608 clones and corresponds to a 36 matrix pools. Thus, the screening of the entire library could be accomplished in less than 80 PCR reactions including positive and negative controls. As a demonstration of the feasibility of the concept, a set of 24 SSR markers covering about 36 cM in the sunflower SSR map (Tang et al. in Theor Appl Genet 105:1124-1136, 2002) have been used to screen the BAC library. About 125 BAC clones have been identified and then organized in 23 contigs by HindIII digestion. The contigs are anchored on the SSR map and thus constitutes a first-generation physical map of this region. The utility of this BAC library as a genomic resource for physical mapping and map-based cloning in sunflower is discussed