Integrated Molecular and Bioinformatics Approaches for Disease-Related Genes in Plants.

Modern plant pathology relies on bioinformatics approaches to create novel plant disease diagnostic tools. In recent years, a significant amount of biological data has been generated due to rapid developments in genomics and molecular biology techniques. The progress in the sequencing of agriculturally important crops has made it possible to develop a better understanding of plant-pathogen interactions and plant resistance. The availability of host-pathogen genome data offers effective assistance in retrieving, annotating, analyzing, and identifying the functional aspects for characterization at the gene and genome levels. Physical mapping facilitates the identification and isolation of several candidate resistance (R) genes from diverse plant species. A large number of genetic variations, such as disease-causing mutations in the genome, have been identified and characterized using bioinformatics tools, and these desirable mutations were exploited to develop disease resistance. Moreover, crop genome editing tools, namely the CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas9 (CRISPR-associated) system, offer novel and efficient strategies for developing durable resistance. This review paper describes some aspects concerning the databases, tools, and techniques used to characterize resistance (R) genes for plant disease management.

Approaching the mapping limit with closed-loop mapping strategy for deploying neural networks on neuromorphic hardware.

The decentralized manycore architecture is broadly adopted by neuromorphic chips for its high computing parallelism and memory locality. However, the fragmented memories and decentralized execution make it hard to deploy neural network models onto neuromorphic hardware with high resource utilization and processing efficiency. There are usually two stages during the model deployment: one is the logical mapping that partitions parameters and computations into small slices and allocate each slice into a single core with limited resources; the other is the physical mapping that places each logical core to a physical location in the chip. In this work, we propose the mapping limit concept for the first time that points out the resource saving upper limit in logical and physical mapping. Furthermore, we propose a closed-loop mapping strategy with an asynchronous 4D model partition for logical mapping and a Hamilton loop algorithm (HLA) for physical mapping. We implement the mapping methods on our state-of-the-art neuromorphic chip, TianjicX. Extensive experiments demonstrate the superior performance of our mapping methods, which can not only outperform existing methods but also approach the mapping limit. We believe the mapping limit concept and the closed-loop mapping strategy can help build a general and efficient mapping framework for neuromorphic hardware.

Extended DNA Fibers for High-Resolution Mapping.

DNA fiber-FISH is an easy and simple light microscopic method to map unique and repeat sequences relative to each other at the molecular scale. A standard fluorescence microscope and a DNA labeling kit are sufficient to visualize DNA sequences from any tissue or organ. Despite the enormous progress of high-throughput sequencing technologies, DNA fiber-FISH remains a unique and indispensable tool to detect chromosomal rearrangements and to demonstrate differences between related species at high resolution. We discuss standard and alternative steps to easily prepare extended DNA fibers for high-resolution FISH mapping.

Physical mapping of a new powdery mildew resistance locus from Thinopyrum ponticum chromosome 4AgS.

Thinopyrum ponticum (Podp.) Barkworth and D.R. Dewey is a decaploid species that has served as an important genetic resource for improving wheat for the better part of a century. The wheat-Th. ponticum 4Ag (4D) disomic substitution line Blue 58, which was obtained following the distant hybridization between Th. ponticum and common wheat, has been stably resistant to powdery mildew under field conditions for more than 40 years. The transfer of 4Ag into the susceptible wheat cultivar Xiaoyan 81 resulted in powdery mildew resistance, indicating the alien chromosome includes the resistance locus. Irradiated Blue 58 pollen were used for the pollination of the recurrent parent Xiaoyan 81, which led to the development of four stable wheat-Th. ponticum 4Ag translocation lines with diverse alien chromosomal segments. The assessment of powdery mildew resistance showed that translocation line L1 was susceptible, but the other three translocation lines (WTT139, WTT146, and WTT323) were highly resistant. The alignment of 81 specific-locus amplified fragments to the Th. elongatum genome revealed that 4Ag originated from a group 4 chromosome. The corresponding physical positions of every 4Ag-derived fragment were determined according to a cytogenetic analysis, the amplification of specific markers, and a sequence alignment. Considering the results of the evaluation of disease resistance, the Pm locus was mapped to the 3.79-97.12 Mb region of the short arm of chromosome 4Ag. Because of its durability, this newly identified Pm locus from a group 4 chromosome of Th. ponticum may be important for breeding wheat varieties with broad-spectrum disease resistance.

Phenotypic characterization of the wheat temperature-sensitive leaf color mutant and physical mapping of mutant gene by reduced-representation sequencing.

Few available leaf color mutants in crops have greatly limited the understanding of photosynthesis mechanisms, leading to few accomplishments in crop yield improvement via enhanced photosynthetic efficiency. Here, a noticeable albino mutant, CN19M06, was identified. A comparison between CN19M06 and the wild type CN19 at different temperatures showed that the albino mutant was temperature-sensitive and produced leaves with a decreased chlorophyll content at temperatures below 10 °C. Genetic analysis suggested that the albinism was controlled by one recessive nuclear gene named TSCA1, which was putatively assigned to the region of 718.1-729.8 Mb on chromosome 2AL using bulked-segregant analysis and double-digest restriction site-associated DNA. Finally, molecular linkage analysis physically anchored TSCA1 to a narrowed region of 718.8-725.3 Mb with a 6.5 Mb length on 2AL flanked by InDel 18 and InDel 25 with 0.7 cM genetic interval. Among the 111 annotated functional genes in the corresponding chromosomal region, only TraesCS2A01G487900 of the PAP fibrillin family was both related to chlorophyll metabolism and temperature sensitivity; therefore, it was considered the putative candidate gene of TSCA1. Overall, CN19M06 has great potential for exploring the molecular mechanism of photosynthesis and monitoring temperature changes in wheat production.

Identification of a Small Translocation from 6R Possessing Stripe Rust Resistance to Wheat.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici Eriks. & E. Henn, is the most devastating fungal disease of bread wheat. Here, a wheat-rye multiple disomic substitution line, SLU126 4R (4D), 5R (5D), and 6R (7D), possessing resistance against 25 races of P. striiformis f. sp. tritici, was used and crossed with Chinese Spring ph1b to induce homeologous recombination to produce introgressions with a reduced rye chromosome segment. Seedling assays confirmed that the stripe rust resistance from SLU126 was retained over multiple generations. Through genotyping-by-sequencing (GBS) platforms and aligning the putative GBS-single-nucleotide polymorphism (SNPs) to the full-length annotated rye nucleotide-binding leucine-rich repeat (NLR) genes in the parental lines (CS ph1b, SLU126, CSA, and SLU820), we identified the physical position of 26, 13, and 9 NLR genes on chromosomes 6R, 4R, and 5R, respectively. The physical positions of 25 NLR genes on chromosome 6R were identified from 568,460,437 bp to 879,958,268 bp in the 6RL chromosome segment. Based on these NLR positions on the 6RL chromosome segment, the three linked SNPs (868,123,650 to 873,285,112 bp) were validated through kompetitive allele-specific PCR (KASP) assays in SLU126 and resistance plants in the family 29-N3-5. Using these KASP markers, we identified a small piece of the rye translocation (i.e., as a possible 6DS.6DL.6RL.6DL) containing the stripe resistance gene, temporary designated YrSLU, within the 6RL segment. This new stripe rust resistance gene provides an additional asset for wheat improvement to mitigate yield losses caused by stripe rust.

Development and Characterization of Triticum aestivum-Aegilops longissima 6Sl Recombinants Harboring a Novel Powdery Mildew Resistance Gene Pm6Sl.

Powdery mildew of wheat is a foliar disease that is spread worldwide. Cultivation of resistant varieties is the most effective, economical, and environmentally friendly strategy to curb this disease. Powdery mildew resistance genes (Pm) are the primary resources for resistance breeding, and new Pm genes are in constant demand. Previously, we identified Aegilops longissima chromosome 6Sl#3 as a carrier of powdery mildew resistance and designated the resistance gene as Pm6Sl. Here, we reported the design of 24 markers specific to 6Sl#3 on the basis of the full-length cDNA sequences of 6Sl#3 donor Ae. longissma accession TA1910, and the development of wheat-Ae. longissima 6Sl#3 introgression stocks by ph1b-induced homoeologous recombination. Further, 6Sl#3 introgression lines were identified and characterized by integration analysis of powdery mildew responses, in situ hybridization, and molecular markers and Pm6Sl was mapped to a distal interval of 42.80 Mb between markers Ael58410 and Ael57699 in the long arm of 6Sl#3. Two resistant recombinants, R43 (T6BS.6BL-6Sl#3L) and T27 (Ti6AS.6AL-6Sl#3L-6AL), contained segments harboring Pm6Sl with less than 8% of 6Sl#3 genomic length, and two markers were diagnostic for Pm6Sl. This study broadened powdery mildew resistance gene resources for wheat improvement and provided a fundamental basis for fine mapping and cloning of Pm6Sl to further understand its molecular mechanism of disease resistance.

Study and Physical Mapping of the Species-Specific Tandem Repeat CS-237 Linked with 45S Ribosomal DNA Intergenic Spacer in Cannabis sativa L.

Hemp (Cannabis sativa L.) is a valuable crop and model plant for studying sex chromosomes. The scientific interest in the plant has led to its whole genome sequencing and the determination of its cytogenetic characteristics. A range of cytogenetic markers (subtelomeric repeat CS-1, 5S rDNA, and 45S rDNA) has been mapped onto hemp's chromosomes by fluorescent in situ hybridization (FISH). In this study, another cytogenetic marker (the tandem repeat CS-237, with a 237 bp monomer) was found, studied, and localized on chromosomes by FISH. The signal distribution and karyotyping revealed that the CS-237 probe was localized in chromosome 6 with one hybridization site and in chromosome 8 with two hybridization sites, one of which colocalizes with the 45S rDNA probe (with which a nucleolus organizer region, NOR, was detected). A BLAST analysis of the genomic data and PCR experiments showed that the modified CS-237 monomers (delCS-237, 208 bp in size) were present in the intergenic spacers (IGSs) of hemp 45S rDNA monomers. Such a feature was firstly observed in Cannabaceae species. However, IGS-linked DNA repeats were found in several plant species of other families (Fabaceae, Solanaceae, and Asteraceae). This phenomenon is discussed in this article. The example of CS-237 may be useful for further studying the phenomenon as well as for the physical mapping of hemp chromosomes.

A Gene-Based Method for Cytogenetic Mapping of Repeat-Rich Mosquito Genomes.

Long-read sequencing technologies have opened up new avenues of research on the mosquito genome biology, enabling scientists to better understand the remarkable abilities of vectors for transmitting pathogens. Although new genome mapping technologies such as Hi-C scaffolding and optical mapping may significantly improve the quality of genomes, only cytogenetic mapping, with the help of fluorescence in situ hybridization (FISH), connects genomic scaffolds to a particular chromosome and chromosome band. This mapping approach is important for creating and validating chromosome-scale genome assemblies for mosquitoes with repeat-rich genomes, which can potentially be misassembled. In this study, we describe a new gene-based physical mapping approach that was optimized using the newly assembled Aedes albopictus genome, which is enriched with transposable elements. To avoid amplification of the repetitive DNA, 15 protein-coding gene transcripts were used for the probe design. Instead of using genomic DNA, complementary DNA was utilized as a template for development of the PCR-amplified probes for FISH. All probes were successfully amplified and mapped to specific chromosome bands. The genome-unique probes allowed to perform unambiguous mapping of genomic scaffolds to chromosome regions. The method described in detail here can be used for physical genome mapping in other insects.

Evolutionary insights into the genomic organization of major ribosomal DNA in ant chromosomes.

The major rDNA genes are composed of tandem repeats and are part of the nucleolus organizing regions (NORs). They are highly conserved and therefore useful in understanding the evolutionary patterns of chromosomal locations. The evolutionary dynamics of the karyotype may affect the organization of rDNA genes within chromosomes. In this study, we physically mapped 18S rDNA genes in 13 Neotropical ant species from four subfamilies using fluorescence in situ hybridization. Furthermore, a survey of published rDNA cytogenetic data for 50 additional species was performed, which allowed us to detect the evolutionary patterns of these genes in ant chromosomes. Species from the Neotropical, Palearctic, and Australian regions, comprising a total of 63 species from 19 genera within six subfamilies, were analysed. Most of the species (48 out of 63) had rDNA genes restricted to a single chromosome pair in their intrachromosomal regions. The position of rDNA genes within the chromosomes appears to hinder their dispersal throughout the genome, as translocations and ectopic recombination are uncommon in intrachromosomal regions because they can generate meiotic abnormalities. Therefore, rDNA genes restricted to a single chromosome pair seem to be a plesiomorphic feature in ants, while multiple rDNA sites, observed in distinct subfamilies, may have independent origins in different genera.

Physical Mapping of Pm57, a Powdery Mildew Resistance Gene Derived from Aegilops searsii.

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is one of many severe diseases that threaten bread wheat (Triticum aestivum L.) yield and quality worldwide. The discovery and deployment of powdery mildew resistance genes (Pm) can prevent this disease epidemic in wheat. In a previous study, we transferred the powdery mildew resistance gene Pm57 from Aegilops searsii into common wheat and cytogenetically mapped the gene in a chromosome region with the fraction length (FL) 0.75-0.87, which represents 12% segment of the long arm of chromosome 2Ss#1. In this study, we performed RNA-seq using RNA extracted from leaf samples of three infected and mock-infected wheat-Ae. searsii 2Ss#1 introgression lines at 0, 12, 24, and 48 h after inoculation with Bgt isolates. Then we designed 79 molecular markers based on transcriptome sequences and physically mapped them to Ae. searsii chromosome 2Ss#1- in seven intervals. We used these markers to identify 46 wheat-Ae. searsii 2Ss#1 recombinants induced by ph1b, a deletion mutant of pairing homologous (Ph) genes. After analyzing the 46 ph1b-induced 2Ss#1L recombinants in the region where Pm57 is located with different Bgt-responses, we physically mapped Pm57 gene on the long arm of 2Ss#1 in a 5.13 Mb genomic region, which was flanked by markers X67593 (773.72 Mb) and X62492 (778.85 Mb). By comparative synteny analysis of the corresponding region on chromosome 2B in Chinese Spring (T. aestivum L.) with other model species, we identified ten genes that are putative plant defense-related (R) genes which includes six coiled-coil nucleotide-binding site-leucine-rich repeat (CNL), three nucleotide-binding site-leucine-rich repeat (NL) and a leucine-rich receptor-like repeat (RLP) encoding proteins. This study will lay a foundation for cloning of Pm57, and benefit the understanding of interactions between resistance genes of wheat and powdery mildew pathogens.

Evolutionary superscaffolding and chromosome anchoring to improve Anopheles genome assemblies.

BACKGROUND: New sequencing technologies have lowered financial barriers to whole genome sequencing, but resulting assemblies are often fragmented and far from 'finished'. Updating multi-scaffold drafts to chromosome-level status can be achieved through experimental mapping or re-sequencing efforts. Avoiding the costs associated with such approaches, comparative genomic analysis of gene order conservation (synteny) to predict scaffold neighbours (adjacencies) offers a potentially useful complementary method for improving draft assemblies. RESULTS: We evaluated and employed 3 gene synteny-based methods applied to 21 Anopheles mosquito assemblies to produce consensus sets of scaffold adjacencies. For subsets of the assemblies, we integrated these with additional supporting data to confirm and complement the synteny-based adjacencies: 6 with physical mapping data that anchor scaffolds to chromosome locations, 13 with paired-end RNA sequencing (RNAseq) data, and 3 with new assemblies based on re-scaffolding or long-read data. Our combined analyses produced 20 new superscaffolded assemblies with improved contiguities: 7 for which assignments of non-anchored scaffolds to chromosome arms span more than 75% of the assemblies, and a further 7 with chromosome anchoring including an 88% anchored Anopheles arabiensis assembly and, respectively, 73% and 84% anchored assemblies with comprehensively updated cytogenetic photomaps for Anopheles funestus and Anopheles stephensi. CONCLUSIONS: Experimental data from probe mapping, RNAseq, or long-read technologies, where available, all contribute to successful upgrading of draft assemblies. Our evaluations show that gene synteny-based computational methods represent a valuable alternative or complementary approach. Our improved Anopheles reference assemblies highlight the utility of applying comparative genomics approaches to improve community genomic resources.

The improved assembly of 7DL chromosome provides insight into the structure and evolution of bread wheat.

Wheat is one of the most important staple crops worldwide and also an excellent model species for crop evolution and polyploidization studies. The breakthrough of sequencing the bread wheat genome and progenitor genomes lays the foundation to decipher the complexity of wheat origin and evolutionary process as well as the genetic consequences of polyploidization. In this study, we sequenced 3286 BACs from chromosome 7DL of bread wheat cv. Chinese Spring and integrated the unmapped contigs from IWGSC v1 and available PacBio sequences to close gaps present in the 7DL assembly. In total, 8043 out of 12 825 gaps, representing 3 491 264 bp, were closed. We then used the improved assembly of 7DL to perform comparative genomic analysis of bread wheat (Ta7DL) and its D donor, Aegilops tauschii (At7DL), to identify domestication signatures. Results showed a strong syntenic relationship between Ta7DL and At7DL, although some small rearrangements were detected at the distal regions. A total of 53 genes appear to be lost genes during wheat polyploidization, with 23% (12 genes) as RGA (disease resistance gene analogue). Furthermore, 86 positively selected genes (PSGs) were identified, considered to be domestication-related candidates. Finally, overlapping of QTLs obtained from GWAS analysis and PSGs indicated that TraesCS7D02G321000 may be one of the domestication genes involved in grain morphology. This study provides comparative information on the sequence, structure and organization between bread wheat and Ae. tauschii from the perspective of the 7DL chromosome, which contribute to better understanding of the evolution of wheat, and supports wheat crop improvement.

Gene Duplication in the Sugarcane Genome: A Case Study of Allele Interactions and Evolutionary Patterns in Two Genic Regions.

Sugarcane (Saccharum spp.) is highly polyploid and aneuploid. Modern cultivars are derived from hybridization between S. officinarum and S. spontaneum. This combination results in a genome exhibiting variable ploidy among different loci, a huge genome size (~10 Gb) and a high content of repetitive regions. An approach using genomic, transcriptomic, and genetic mapping can improve our knowledge of the behavior of genetics in sugarcane. The hypothetical HP600 and Centromere Protein C (CENP-C) genes from sugarcane were used to elucidate the allelic expression and genomic and genetic behaviors of this complex polyploid. The physically linked side-by-side genes HP600 and CENP-C were found in two different homeologous chromosome groups with ploidies of eight and ten. The first region (Region01) was a Sorghum bicolor ortholog region with all haplotypes of HP600 and CENP-C expressed, but HP600 exhibited an unbalanced haplotype expression. The second region (Region02) was a scrambled sugarcane sequence formed from different noncollinear genes containing partial duplications of HP600 and CENP-C (paralogs). This duplication resulted in a non-expressed HP600 pseudogene and a recombined fusion version of CENP-C and the orthologous gene Sobic.003G299500 with at least two chimeric gene haplotypes expressed. It was also determined that it occurred before Saccharum genus formation and after the separation of sorghum and sugarcane. A linkage map was constructed using markers from nonduplicated Region01 and for the duplication (Region01 and Region02). We compare the physical and linkage maps, demonstrating the possibility of mapping markers located in duplicated regions with markers in nonduplicated region. Our results contribute directly to the improvement of linkage mapping in complex polyploids and improve the integration of physical and genetic data for sugarcane breeding programs. Thus, we describe the complexity involved in sugarcane genetics and genomics and allelic dynamics, which can be useful for understanding complex polyploid genomes.

Physical Mapping of Peroxidase Genes and Development of Functional Markers for TaPod-D1 on Bread Wheat Chromosome 7D.

Peroxidase (POD) activity in wheat (Triticum aestivum L.) grain influences natural carotenoid pigment content and is associated with the color of flour, and processing and product quality. Here, we report the molecular characterization and physical mapping of POD genes in bread wheat. The complete genomic DNA (gDNA) sequence of two POD genes (TaPod-A2 and TaPod-D1), and the partial gDNA sequence of two additional POD genes (TaPod-A3 and TaPod-B1) from wheat were characterized using in silico cloning and validated through laboratory experiments. Using a set of 21 nullisomic-tetrasomic (NT) lines, six group-7 ditelosomic (Dt) lines, and 38 group-7 deletion (Del) lines of Chinese Spring (CS), TaPod-A2 and TaPod-D1 were found to be physically located on 0.73-0.83 and on the most distal 0.39 fraction arm length (FL) of 7AS and 7DS in cv. CS, respectively; whereas, TaPod-A3 and TaPod-B1 were assigned to the 0.40-0.49 and 0.40-0.48 FL of 7AL and 7BL, respectively. Based on single nucleotide polymorphisms (SNPs) of two alleles at the TaPod-D1 locus, two functional markers POD-7D1 and POD-7D6 were developed, amplifying 540- and 640-bp, fragments in varieties with higher and lower POD activities, respectively. A total of 224 wheat varieties were analyzed and showed a significant association between the polymorphic fragments and POD activity using POD-7D1 and POD-7D6 markers. The analysis of variance (ANOVA) indicated the average POD activities of 115 varieties with TaPod-D1a were significantly lower than 109 varieties with TaPod-D1b (P < 0.01). This study provides useful information of the POD genes in bread wheat, insight into wheat genome synteny and structure, gene-specific markers, and contributes a valuable resource for quality improvement in wheat breeding programs.

Sunflower resistance to multiple downy mildew pathotypes revealed by recognition of conserved effectors of the oomycete Plasmopara halstedii.

Over the last 40 years, new sunflower downy mildew isolates (Plasmopara halstedii) have overcome major gene resistances in sunflower, requiring the identification of additional and possibly more durable broad-spectrum resistances. Here, 354 RXLR effectors defined in silico from our new genomic data were classified in a network of 40 connected components sharing conserved protein domains. Among 205 RXLR effector genes encoding conserved proteins in 17 P. halstedii pathotypes of varying virulence, we selected 30 effectors that were expressed during plant infection as potentially essential genes to target broad-spectrum resistance in sunflower. The transient expression of the 30 core effectors in sunflower and in Nicotiana benthamiana leaves revealed a wide diversity of targeted subcellular compartments, including organelles not so far shown to be targeted by oomycete effectors such as chloroplasts and processing bodies. More than half of the 30 core effectors were able to suppress pattern-triggered immunity in N. benthamiana, and five of these induced hypersensitive responses (HR) in sunflower broad-spectrum resistant lines. HR triggered by PhRXLRC01 co-segregated with Pl22 resistance in F3 populations and both traits localized in 1.7 Mb on chromosome 13 of the sunflower genome. Pl22 resistance was physically mapped on the sunflower genome recently sequenced, unlike all the other downy mildew resistances published so far. PhRXLRC01 and Pl22 are proposed as an avirulence/resistance gene couple not previously described in sunflower. Core effector recognition is a successful strategy to accelerate broad-spectrum resistance gene identification in complex crop genomes such as sunflower.

Stable Quantitative Resistance Loci to Blackleg Disease in Canola (Brassica napus L.) Over Continents.

The hemibiotrophic fungus, Leptosphaeria maculans is the most devastating pathogen, causing blackleg disease in canola (Brassica napus L). To study the genomic regions involved in quantitative resistance (QR), 259-276 DH lines from Darmor-bzh/Yudal (DYDH) population were assessed for resistance to blackleg under shade house and field conditions across 3 years. In different experiments, the broad sense heritability varied from 43 to 95%. A total of 27 significant quantitative trait loci (QTL) for QR were detected on 12 chromosomes and explained between 2.14 and 10.13% of the genotypic variance. Of the significant QTL, at least seven were repeatedly detected across different experiments on chromosomes A02, A07, A09, A10, C01, and C09. Resistance alleles were mainly contributed by 'Darmor-bzh' but 'Yudal' also contributed few of them. Our results suggest that plant maturity and plant height may have a pleiotropic effect on QR in our conditions. We confirmed that Rlm9 which is present in 'Darmor-bzh' is not effective to confer resistance in our Australian field conditions. Comparative mapping showed that several R genes coding for nucleotide-binding leucine-rich repeat (LRR) receptors map in close proximity (within 200 Kb) of the significant trait-marker associations on the reference 'Darmor-bzh' genome assembly. More importantly, eight significant QTL regions were detected across diverse growing environments: Australia, France, and United Kingdom. These stable QTL identified herein can be utilized for enhancing QR in elite canola germplasm via marker- assisted or genomic selection strategies.

The Bermuda Triangle: The Pragmatics, Policies, and Principles for Data Sharing in the History of the Human Genome Project.

The Bermuda Principles for DNA sequence data sharing are an enduring legacy of the Human Genome Project (HGP). They were adopted by the HGP at a strategy meeting in Bermuda in February of 1996 and implemented in formal policies by early 1998, mandating daily release of HGP-funded DNA sequences into the public domain. The idea of daily sharing, we argue, emanated directly from strategies for large, goal-directed molecular biology projects first tested within the "community" of C. elegans researchers, and were introduced and defended for the HGP by the nematode biologists John Sulston and Robert Waterston. In the C. elegans community, and subsequently in the HGP, daily sharing served the pragmatic goals of quality control and project coordination. Yet in the HGP human genome, we also argue, the Bermuda Principles addressed concerns about gene patents impeding scientific advancement, and were aspirational and flexible in implementation and justification. They endured as an archetype for how rapid data sharing could be realized and rationalized, and permitted adaptation to the needs of various scientific communities. Yet in addition to the support of Sulston and Waterston, their adoption also depended on the clout of administrators at the US National Institutes of Health (NIH) and the UK nonprofit charity the Wellcome Trust, which together funded 90% of the HGP human sequencing effort. The other nations wishing to remain in the HGP consortium had to accommodate to the Bermuda Principles, requiring exceptions from incompatible existing or pending data access policies for publicly funded research in Germany, Japan, and France. We begin this story in 1963, with the biologist Sydney Brenner's proposal for a nematode research program at the Laboratory of Molecular Biology (LMB) at the University of Cambridge. We continue through 2003, with the completion of the HGP human reference genome, and conclude with observations about policy and the historiography of molecular biology.

Sequencing through thick and thin: Historiographical and philosophical implications.

DNA sequencing has been characterised by scholars and life scientists as an example of 'big', 'fast' and 'automated' science in biology. This paper argues, however, that these characterisations are a product of a particular interpretation of what sequencing is, what I call 'thin sequencing'. The 'thin sequencing' perspective focuses on the determination of the order of bases in a particular stretch of DNA. Based upon my research on the pig genome mapping and sequencing projects, I provide an alternative 'thick sequencing' perspective, which also includes a number of practices that enable the sequence to travel across and be used in wider communities. If we take sequencing in the thin manner to be an event demarcated by the determination of sequences in automated sequencing machines and computers, this has consequences for the historical analysis of sequencing projects, as it focuses attention on those parts of the work of sequencing that are more centralised, fast (and accelerating) and automated. I argue instead that sequencing can be interpreted as a more open-ended process including activities such as the generation of a minimum tile path or annotation, and detail the historiographical and philosophical consequences of this move.

Genetic insight and mapping of the pod constriction trait in Virginia-type peanut.

BACKGROUND: Pod constriction is an important descriptive and agronomic trait of peanut. For the in-shell Virginia marketing-type, this trait has commercial importance as well, since deeply constricted pods have a tendency to break, which makes them unmarketable. Classical genetic studies have indicated that pod constriction in peanut is controlled by one to four genes, depending on the genetic background. In all of those studies, pod constriction was evaluated visually as opposed to quantitatively. Here, we examined the genetic nature of this trait in the Virginia-type background. Our study involved 195 recombinant inbred lines (F7RILs) derived from two closely related cultivars that differ in their degree of pod constriction. Pod constriction was evaluated visually and quantitatively in terms of the pod constriction index (PCI), calculated as the average ratio between the pod's waist and shoulders. RESULTS: ANOVA and genetic parameters for PCI among the F7RILs in three blocks showed very significant genotypic effect (p(F) < 0.0001) and high heritability and genetic gain estimates (0.84 and 0.52, respectively). The mean PCI values of the different RILs had a bimodal distribution with an approximate 1:1 ratio between the two curves. Pod constriction was also determined visually (VPC) by grading the degree of each RIL as 'deep' or 'slight'. The χ2 test was found to not be significantly different from a 1:1 ratio (p = 0.79) as well. SNP-array-based technology was used to map this trait in the RIL population. A major locus for the pod constriction trait was found on chromosome B7, between B07_120,287,958 and B07_120,699,791, and the best-linked SNP explained 32% of the total variation within that region. Some discrepancy was found between the SNPs original location and the genetic mapping of the trait. CONCLUSION: The trait distribution and mapping, together with data from F1 and F2 generations indicate that in this background the pod constriction is controlled by a major recessive gene. The identity of loci controlling the pod constriction trait will allow breeders to apply marker-assisted breeding approaches to shift allelic frequencies towards a slighter pod constriction and will facilitate future effort for map-based gene cloning.