Transcriptome Sequencing of Hevea brasiliensis for Development of Microsatellite Markers and Construction of a Genetic Linkage Map

To obtain more information on the Hevea brasiliensis genome, we sequenced the transcriptome from the vegetative shoot apex yielding 2 311 497 reads. Clustering and assembly of the reads produced a total of 113 313 unique sequences, comprising 28 387 isotigs and 84 926 singletons. Also, 17 819 expressed sequence tag (EST)-simple sequence repeats (SSRs) were identified from the data set. To demonstrate the use of this EST resource for marker development, primers were designed for 430 of the EST-SSRs. Three hundred and twenty-three primer pairs were amplifiable in H. brasiliensis clones. Polymorphic information content values of selected 47 SSRs among 20 H. brasiliensis clones ranged from 0.13 to 0.71, with an average of 0.51. A dendrogram of genetic similarities between the 20 H. brasiliensis clones using these 47 EST-SSRs suggested two distinct groups that correlated well with clone pedigree. These novel EST-SSRs together with the published SSRs were used for the construction of an integrated parental linkage map of H. brasiliensis based on 81 lines of an F1 mapping population. The map consisted of 97 loci, consisting of 37 novel EST-SSRs and 60 published SSRs, distributed on 23 linkage groups and covered 842.9 cM with a mean interval of 11.9 cM and ∼4 loci per linkage group. Although the numbers of linkage groups exceed the haploid number (18), but with several common markers between homologous linkage groups with the previous map indicated that the F1 map in this study is appropriate for further study in marker-assisted selection

A genome scan for quantitative trait loci affecting cyanogenic potential of cassava root in an outbred population

<p>Abstract</p> <p>Background</p> <p>Cassava (<it>Manihot esculenta </it>Crantz) can produce cyanide, a toxic compound, without self-injury. That ability was called the cyanogenic potential (CN). This project aimed to identify quantitative trait loci (QTL) associated with the CN in an outbred population derived from 'Hanatee' × 'Huay Bong 60', two contrasting cultivars. CN was evaluated in 2008 and in 2009 at Rayong province, and in 2009 at Lop Buri province, Thailand. CN was measured using a picrate paper kit. QTL analysis affecting CN was performed with 303 SSR markers.</p> <p>Results</p> <p>The phenotypic values showed continuous variation with transgressive segregation events with more (115 ppm) and less CN (15 ppm) than either parent ('Hanatee' had 33 ppm and 'Huay Bong 60' had 95 ppm). The linkage map consisted of 303 SSR markers, on 27 linkage groups with a map that encompassed 1,328 cM. The average marker interval was 5.8 cM. Five QTL underlying CN were detected. <it>CN08R1</it>from 2008 at Rayong, <it>CN09R1</it>and <it>CN09R2 </it>from 2009 at Rayong, and <it>CN09L1 </it>and <it>CN09L2 </it>from 2009 at Lop Buri were mapped on linkage group 2, 5, 10 and 11, respectively. Among all the identified QTL, <it>CN09R1 </it>was the most significantly associated with the CN trait with LOD score 5.75 and explained the greatest percentage of phenotypic variation (%Expl.) of 26%.</p> <p>Conclusions</p> <p>Five new QTL affecting CN were successfully identified from 4 linkage groups. Discovery of these QTL can provide useful markers to assist in cassava breeding and studying genes affecting the trait.</p

The Complete Chloroplast Genome Sequence of Date Palm (Phoenix dactylifera L.)

BACKGROUND: Date palm (Phoenix dactylifera L.), a member of Arecaceae family, is one of the three major economically important woody palms--the two other palms being oil palm and coconut tree--and its fruit is a staple food among Middle East and North African nations, as well as many other tropical and subtropical regions. Here we report a complete sequence of the data palm chloroplast (cp) genome based on pyrosequencing. METHODOLOGY/PRINCIPAL FINDINGS: After extracting 369,022 cp sequencing reads from our whole-genome-shotgun data, we put together an assembly and validated it with intensive PCR-based verification, coupled with PCR product sequencing. The date palm cp genome is 158,462 bp in length and has a typical quadripartite structure of the large (LSC, 86,198 bp) and small single-copy (SSC, 17,712 bp) regions separated by a pair of inverted repeats (IRs, 27,276 bp). Similar to what has been found among most angiosperms, the date palm cp genome harbors 112 unique genes and 19 duplicated fragments in the IR regions. The junctions between LSC/IRs and SSC/IRs show different features of sequence expansion in evolution. We identified 78 SNPs as major intravarietal polymorphisms within the population of a specific cp genome, most of which were located in genes with vital functions. Based on RNA-sequencing data, we also found 18 polycistronic transcription units and three highly expression-biased genes--atpF, trnA-UGC, and rrn23. CONCLUSIONS: Unlike most monocots, date palm has a typical cp genome similar to that of tobacco--with little rearrangement and gene loss or gain. High-throughput sequencing technology facilitates the identification of intravarietal variations in cp genomes among different cultivars. Moreover, transcriptomic analysis of cp genes provides clues for uncovering regulatory mechanisms of transcription and translation in chloroplasts

diArk 2.0 provides detailed analyses of the ever increasing eukaryotic genome sequencing data

<p>Abstract</p> <p>Background</p> <p>Nowadays, the sequencing of even the largest mammalian genomes has become a question of days with current next-generation sequencing methods. It comes as no surprise that dozens of genome assemblies are released per months now. Since the number of next-generation sequencing machines increases worldwide and new major sequencing plans are announced, a further increase in the speed of releasing genome assemblies is expected. Thus it becomes increasingly important to get an overview as well as detailed information about available sequenced genomes. The different sequencing and assembly methods have specific characteristics that need to be known to evaluate the various genome assemblies before performing subsequent analyses.</p> <p>Results</p> <p>diArk has been developed to provide fast and easy access to all sequenced eukaryotic genomes worldwide. Currently, diArk 2.0 contains information about more than 880 species and more than 2350 genome assembly files. Many meta-data like sequencing and read-assembly methods, sequencing coverage, GC-content, extended lists of alternatively used scientific names and common species names, and various kinds of statistics are provided. To intuitively approach the data the web interface makes extensive usage of modern web techniques. A number of search modules and result views facilitate finding and judging the data of interest. Subscribing to the RSS feed is the easiest way to stay up-to-date with the latest genome data.</p> <p>Conclusions</p> <p>diArk 2.0 is the most up-to-date database of sequenced eukaryotic genomes compared to databases like GOLD, NCBI Genome, NHGRI, and ISC. It is different in that only those projects are stored for which genome assembly data or considerable amounts of cDNA data are available. Projects in planning stage or in the process of being sequenced are not included. The user can easily search through the provided data and directly access the genome assembly files of the sequenced genome of interest. diArk 2.0 is available at <url>http://www.diark.org</url>.</p

The Cassava Genome: Current Progress, Future Directions

The starchy swollen roots of cassava provide an essential food source for nearly a billion people, as well as possibilities for bioenergy, yet improvements to nutritional content and resistance to threatening diseases are currently impeded. A 454-based whole genome shotgun sequence has been assembled, which covers 69% of the predicted genome size and 96% of protein-coding gene space, with genome finishing underway. The predicted 30,666 genes and 3,485 alternate splice forms are supported by 1.4 M expressed sequence tags (ESTs). Maps based on simple sequence repeat (SSR)-, and EST-derived single nucleotide polymorphisms (SNPs) already exist. Thanks to the genome sequence, a high-density linkage map is currently being developed from a cross between two diverse cassava cultivars: one susceptible to cassava brown streak disease; the other resistant. An efficient genotyping-by-sequencing (GBS) approach is being developed to catalog SNPs both within the mapping population and among diverse African farmer-preferred varieties of cassava. These resources will accelerate marker-assisted breeding programs, allowing improvements in disease-resistance and nutrition, and will help us understand the genetic basis for disease resistance

The Chloroplast Genome Sequence of Mungbean (Vigna radiata) Determined by High-throughput Pyrosequencing: Structural Organization and Phylogenetic Relationships

Mungbean is an economically important crop which is grown principally for its protein-rich dry seeds. However, genomic research of mungbean has lagged behind other species in the Fabaceae family. Here, we reported the complete chloroplast (cp) genome sequence of mungbean obtained by the 454 pyrosequencing technology. The mungbean cp genome is 151 271 bp in length which includes a pair of inverted repeats (IRs) of 26 474 bp separated by a small single-copy region of 17 427 bp and a large single-copy region of 80 896 bp. The genome contains 108 unique genes and 19 of these genes are duplicated in the IR. Of these, 75 are predicted protein-coding genes, 4 ribosomal RNA genes and 29 tRNA genes. Relative to other plant cp genomes, we observed two distinct rearrangements: a 50-kb inversion between accD/rps16 and rbcL/trnK-UUU, and a 78-kb rearrangement between trnH/rpl14 and rps19/rps8. We detected sequence length polymorphism in the cp homopolymeric regions at the intra- and inter-specific levels in the Vigna species. Phylogenetic analysis demonstrated a close relationship between Vigna and Phaseolus in the phaseolinae subtribe and provided a strong support for a monophyletic group of the eurosid I

Complete Chloroplast Genome Sequence of a Major Invasive Species, Crofton Weed (Ageratina adenophora)

Crofton weed (Ageratina adenophora) is one of the most hazardous invasive plant species, which causes serious economic losses and environmental damages worldwide. However, the sequence resource and genome information of A. adenophora are rather limited, making phylogenetic identification and evolutionary studies very difficult. Here, we report the complete sequence of the A. adenophora chloroplast (cp) genome based on Illumina sequencing.The A. adenophora cp genome is 150, 689 bp in length including a small single-copy (SSC) region of 18, 358 bp and a large single-copy (LSC) region of 84, 815 bp separated by a pair of inverted repeats (IRs) of 23, 755 bp. The genome contains 130 unique genes and 18 duplicated in the IR regions, with the gene content and organization similar to other Asteraceae cp genomes. Comparative analysis identified five DNA regions (ndhD-ccsA, psbI-trnS, ndhF-ycf1, ndhI-ndhG and atpA-trnR) containing parsimony-informative characters higher than 2%, which may be potential informative markers for barcoding and phylogenetic analysis. Repeat structure, codon usage and contraction of the IR were also investigated to reveal the pattern of evolution. Phylogenetic analysis demonstrated a sister relationship between A. adenophora and Guizotia abyssinica and supported a monophyly of the Asterales.We have assembled and analyzed the chloroplast genome of A. adenophora in this study, which was the first sequenced plastome in the Eupatorieae tribe. The complete chloroplast genome information is useful for plant phylogenetic and evolutionary studies within this invasive species and also within the Asteraceae family

Identification of SNP and SSR markers in eggplant using RAD tag sequencing

<p>Abstract</p> <p>Background</p> <p>The eggplant (<it>Solanum melongena </it>L.) genome is relatively unexplored, especially compared to those of the other major <it>Solanaceae </it>crops tomato and potato. In particular, no SNP markers are publicly available; on the other hand, over 1,000 SSR markers were developed and publicly available. We have combined the recently developed Restriction-site Associated DNA (RAD) approach with Illumina DNA sequencing for rapid and mass discovery of both SNP and SSR markers for eggplant.</p> <p>Results</p> <p>RAD tags were generated from the genomic DNA of a pair of eggplant mapping parents, and sequenced to produce ~17.5 Mb of sequences arrangeable into ~78,000 contigs. The resulting non-redundant genomic sequence dataset consisted of ~45,000 sequences, of which ~29% were putative coding sequences and ~70% were in common between the mapping parents. The shared sequences allowed the discovery of ~10,000 SNPs and nearly 1,000 indels, equivalent to a SNP frequency of 0.8 per Kb and an indel frequency of 0.07 per Kb. Over 2,000 of the SNPs are likely to be mappable via the Illumina GoldenGate assay. A subset of 384 SNPs was used to successfully fingerprint a panel of eggplant germplasm, producing a set of informative diversity data. The RAD sequences also included nearly 2,000 putative SSRs, and primer pairs were designed to amplify 1,155 loci.</p> <p>Conclusion</p> <p>The high throughput sequencing of the RAD tags allowed the discovery of a large number of DNA markers, which will prove useful for extending our current knowledge of the genome organization of eggplant, for assisting in marker-aided selection and for carrying out comparative genomic analyses within the <it>Solanaceae </it>family.</p

The complete sequence of the Acacia ligulata chloroplast genome reveals a highly divergent clpP1 gene

Legumes are a highly diverse angiosperm family that include many agriculturally important species. To date, 21 complete chloroplast genomes have been sequenced from legume crops confined to the Papilionoideae subfamily. Here we report the first chloroplast genome from the Mimosoideae, Acacia ligulata, and compare it to the previously sequenced legume genomes. The A. ligulata chloroplast genome is 158,724 bp in size, comprising inverted repeats of 25,925 bp and single-copy regions of 88,576 bp and 18,298 bp. Acacia ligulata lacks the inversion present in many of the Papilionoideae, but is not otherwise significantly different in terms of gene and repeat content. The key feature is its highly divergent clpP1 gene, normally considered essential in chloroplast genomes. In A. ligulata, although transcribed and spliced, it probably encodes a catalytically inactive protein. This study provides a significant resource for further genetic research into Acacia and the Mimosoideae. The divergent clpP1 gene suggests that Acacia will provide an interesting source of information on the evolution and functional diversity of the chloroplast Clp protease comple

Exploring the Switchgrass Transcriptome Using Second-Generation Sequencing Technology

Background: Switchgrass (Panicum virgatum L.) is a C4 perennial grass and widely popular as an important bioenergy crop. To accelerate the pace of developing high yielding switchgrass cultivars adapted to diverse environmental niches, the generation of genomic resources for this plant is necessary. The large genome size and polyploid nature of switchgrass makes whole genome sequencing a daunting task even with current technologies. Exploring the transcriptional landscape using next generation sequencing technologies provides a viable alternative to whole genome sequencing in switchgrass. Principal Findings: Switchgrass cDNA libraries from germinating seedlings, emerging tillers, flowers, and dormant seeds were sequenced using Roche 454 GS-FLX Titanium technology, generating 980,000 reads with an average read length of 367 bp. De novo assembly generated 243,600 contigs with an average length of 535 bp. Using the foxtail millet genome as a reference greatly improved the assembly and annotation of switchgrass ESTs. Comparative analysis of the 454-derived switchgrass EST reads with other sequenced monocots including Brachypodium, sorghum, rice and maize indicated a 70– 80 % overlap. RPKM analysis demonstrated unique transcriptional signatures of the four tissues analyzed in this study. More than 24,000 ESTs were identified in the dormant seed library. In silico analysis indicated that there are more than 2000 EST-SSRs in this collection. Expression of several orphan ESTs was confirmed by RT-PCR. Significance: We estimate that about 90 % of the switchgrass gene space has been covered in this analysis. This study nearl