Duangjai Sangsrakru

SSR markers in transcripts of genes linked to post-transcriptional and transcriptional regulatory functions during vegetative and reproductive development of Elaeis guineensis

BackgroundThe oil palm (Elaeis guineensis Jacq.) is a perennial monocotyledonous tropical crop species that is now the worlds number one source of edible vegetable oil, and the richest dietary source of provitamin A. While new elite genotypes from traditional breeding programs provide steady yield increases, the long selection cycle (10-12 years) and the large areas required to cultivate oil palm make genetic improvement slow and labor intensive. Molecular breeding programs have the potential to make significant impacts on the rate of genetic improvement but the limited molecular resources, in particular the lack of molecular markers for agronomic traits of interest, restrict the application of molecular breeding schemes for oil palm.ResultsIn the current study, 6,103 non-redundant ESTs derived from cDNA libraries of developing vegetative and reproductive tissues were annotated and searched for simple sequence repeats (SSRs). Primer pairs from sequences flanking 289 EST-SSRs were tested to detect polymorphisms in elite breeding parents and their crosses. 230 of these amplified PCR products, 88 of which were polymorphic within the breeding material tested. A detailed analysis and annotation of the EST-SSRs revealed the locations of the polymorphisms within the transcripts, and that the main functional category was related to transcription and post-transcriptional regulation. Indeed, SSR polymorphisms were found in sequences encoding AP2-like, bZIP, zinc finger, MADS-box, and NAC-like transcription factors in addition to other transcriptional regulatory proteins and several RNA interacting proteins.ConclusionsThe identification of new EST-SSRs that detect polymorphisms in elite breeding material provides tools for molecular breeding strategies. The identification of SSRs within transcripts, in particular those that encode proteins involved in transcriptional and post-transcriptional regulation, will allow insight into the functional roles of these proteins by studying the phenotypic traits that cosegregate with these markers. Finally, the oil palm EST-SSRs derived from vegetative and reproductive development will be useful for studies on the evolution of the functional diversity within the palm family.

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.

Characterization of microsatellites and gene contents from genome shotgun sequences of mungbean (Vigna radiata (L.) Wilczek)

BackgroundMungbean is an important economical crop in Asia. However, genomic research has lagged behind other crop species due to the lack of polymorphic DNA markers found in this crop. The objective of this work is to develop and characterize microsatellite or simple sequence repeat (SSR) markers from genome shotgun sequencing of mungbean.ResultWe have generated and characterized a total of 470,024 genome shotgun sequences covering 100.5 Mb of the mungbean (Vigna radiata (L.) Wilczek) genome using 454 sequencing technology. We identified 1,493 SSR motifs that could be used as potential molecular markers. Among 192 tested primer pairs in 17 mungbean accessions, 60 loci revealed polymorphism with polymorphic information content (PIC) values ranging from 0.0555 to 0.6907 with an average of 0.2594. Majority of microsatellite markers were transferable in Vigna species, whereas transferability rates were only 22.90% and 24.43% in Phaseolus vulgaris and Glycine max, respectively. We also used 16 SSR loci to evaluate phylogenetic relationship of 35 genotypes of the Asian Vigna group. The genome survey sequences were further analyzed to search for gene content. The evidence suggested 1,542 gene fragments have been sequence tagged, that fell within intersected existing gene models and shared sequence homology with other proteins in the database. Furthermore, potential microRNAs that could regulate developmental stages and environmental responses were discovered from this dataset.ConclusionIn this report, we provided evidence of generating remarkable levels of diverse microsatellite markers and gene content from high throughput genome shotgun sequencing of the mungbean genomic DNA. The markers could be used in germplasm analysis, accessing genetic diversity and linkage mapping of mungbean.

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.

Genome-wide SNP discovery and identification of QTL associated with agronomic traits in oil palm using genotyping-by-sequencing (GBS).

Oil palm has become one of the most important oil crops in the world. Marker-assisted selections have played a pivotal role in oil palm breeding programs. Here, we report the use of genotyping-by-sequencing (GBS) approach for a large-scale SNP discovery and genotyping of a mapping population. Reduced representation libraries of 108 F2 progeny were sequenced and a total of 524 million reads were obtained. We detected 21,471 single nucleotide substitutions, most of which (62.6%) represented transition events. Of 3417 fully informative SNP markers, we were able to place 1085 on a linkage map, which spanned 1429.6 cM and had an average of one marker every 1.26 cM. Three QTL affecting trunk height were detected on LG 10, 14 and 15, whereas a single QTL associated with fruit bunch weight was identified on LG 3. The use of GBS approach proved to be rapid, cost-effective and highly reproducible in this species.

Characterization of the complete chloroplast genome of Hevea brasiliensis reveals genome rearrangement, RNA editing sites and phylogenetic relationships.

Rubber tree (Hevea brasiliensis) is an economical plant and widely grown for natural rubber production. However, genomic research of rubber tree has lagged behind other species in the Euphorbiaceae family. We report the complete chloroplast genome sequence of rubber tree as being 161,191 bp in length including a pair of inverted repeats of 26,810 bp separated by a small single copy region of 18,362 bp and a large single copy region of 89,209 bp. The chloroplast genome contains 112 unique genes, 16 of which are duplicated in the inverted repeat. Of the 112 unique genes, 78 are predicted protein-coding genes, 4 are ribosomal RNA genes and 30 are tRNA genes. Relative to other plant chloroplast genomes, we observed a unique rearrangement in the rubber tree chloroplast genome: a 30-kb inversion between the trnE(UUC)-trnS(GCU) and the trnT(GGU)-trnR(UCU). A comparison between the rubber tree chloroplast genes and cDNA sequences revealed 51 RNA editing sites in which most (48 sites) were located in 26 protein coding genes and the other 3 sites were in introns. Phylogenetic analysis based on chloroplast genes demonstrated a close relationship between Hevea and Manihot in Euphorbiaceae and provided a strong support for a monophyletic group of the eurosid I.

Elucidation of the molecular responses to waterlogging in Jatropha roots by transcriptome profiling

Jatropha (Jatropha curcas) is a promising oil-seed crop for biodiesel production. However, the species is highly sensitive to waterlogging, which can result in stunted growth and yield loss. To date, the molecular mechanisms underlying the responses to waterlogging in Jatropha remain elusive. Here, the transcriptome adjustment of Jatropha roots to waterlogging was examined by high-throughput RNA-sequencing (RNA-seq). The results indicated that 24 h of waterlogging caused significant changes in mRNA abundance of 1968 genes. Comprehensive gene ontology and functional enrichment analysis of root transcriptome revealed that waterlogging promoted responses to hypoxia and anaerobic respiration. On the other hand, the stress inhibited carbohydrate synthesis, cell wall biogenesis, and growth. The results also highlighted the roles of ethylene, nitrate, and nitric oxide in waterlogging acclimation. In addition, transcriptome profiling identified 85 waterlogging-induced transcription factors including members of AP2/ERF, MYB, and WRKY families implying that reprogramming of gene expression is a vital mechanism for waterlogging acclimation. Comparative analysis of differentially regulated transcripts in response to waterlogging among Arabidopsis, gray poplar, Jatropha, and rice further revealed not only conserved but species-specific regulation. Our findings unraveled the molecular responses to waterlogging in Jatropha and provided new perspectives for developing a waterlogging tolerant cultivar in the future.

Transcriptome analysis of normal and mantled developing oil palm flower and fruit

Elaeis guineensis (oil palm) accounts for a large and increasing proportion of the worlds cooking oil production. Cloning via somatic embryogenesis results in a somaclonal variant known as mantled which produce fruit with little to no oil yield. The mantled phenotype is believed to be epigenetic in nature. We performed RNA-Seq on developing flower and fruit samples of normal and mantled oil palm to characterize their transcriptomes. We present expression data for all transcripts in normal and mantled flower and fruit samples. Many genes are differentially expressed, including several from pathways that may be the cause of the mantled phenotype if disrupted, such as genes involved in primary hormone responses, DNA replication and repair, chromatin remodeling and a gene involved in RNA mediated DNA methylation. In addition, the gene expression data for developing flower and fruit will serve as a valuable resource for oil palm genetics and genomic studies.

Draft genome sequence of Arthrospira platensis C1 (PCC9438)

Arthrospira platensis is a cyanobacterium that is extensively cultivated outdoors on a large commercial scale for consumption as a food for humans and animals. It can be grown in monoculture under highly alkaline conditions, making it attractive for industrial production. Here we describe the complete genome sequence of A. platensis C1 strain and its annotation. The A. platensis C1 genome contains 6,089,210 bp including 6,108 protein-coding genes and 45 RNA genes, and no plasmids. The genome information has been used for further comparative analysis, particularly of metabolic pathways, photosynthetic efficiency and barriers to gene transfer.

Single nucleotide polymorphism marker development in the rubber tree, Hevea brasiliensis (Euphorbiaceae)

PREMISE OF THE STUDY We demonstrated the application of high-throughput 454 sequencing technology in the identification of single nucleotide polymorphism (SNP) markers in Hevea brasiliensis. METHODS AND RESULTS A total of 5883 putative SNP positions were discovered in silico, and 10 biallelic SNP markers were validated from 454-derived EST sequences. The polymorphism information content (PIC) and the observed heterozygosity (H(o)) ranged from 0.0963-0.5135 and 0.1071-0.4643, respectively. CONCLUSIONS These markers can be useful for the construction of genetic maps, the identification of quantitative trait loci linked to commercially desirable traits, and the study of genetic structure in H. brasiliensis.