Comparative analysis of expressed sequence tags (ESTs) from Triticum monococcum shoot apical meristem at vegetative and reproductive stages

Triticum monococcum has recently drawn the attention of biologists to discover and utilize novel genes and alleles. To explore the molecular features of the genetic network governing floral transition in shoot apical meristem (SAM) of spring growth habit T. monococcum, two expressed sequence tag (EST) libraries containing 3,031 ESTs from vegetative SAM (VS) and 2,647 ESTs from early reproductive SAM (RS) were analyzed. Assembly of ESTs resulted in 2,303 unigenes for VS library (368 contigs and 1,935 singletons) and 1,890 unigenes (337 contigs and 1,553 singletons) for RS library. The 67.05 % of VS unigenes and 66.30 % of RS unigenes showed significant similarity with genes of known, putative and or unknown function, whereas the remaining 32.95 % of the VS unigenes and 33.7 % of RS unigenes displayed no significant match with the public protein database. The 1,064 and 866 unigenes of VS and RS libraries were assigned to functional categories using Pageman ontology tool. Further analysis revealed that the switch from VS to RS caused significant changes in the abundance of unigenes assigned to some functional categories. A total of 37 genes were identified which were significantly differentially expressed between vegetative and reproductive stages of T. monococcum SAM. Investigation of the differentially expressed genes revealed the importance of the genes involved in energy metabolism, ubiquitin/26S proteasome system, polyamines biosynthesis and signaling of reactive oxygen species in SAM differentiation towards floral transition in T. monococcum.     

Transcriptome generation and analysis from spleen of Indian catfish, Clarias batrachus (Linnaeus, 1758) through normalized cDNA library

Catfishes are commercially important fish for both the fisheries and aquaculture industry. Clarias batrachus, an Indian catfish species is economically important owing to its high demand. A normalized cDNA library was constructed from spleen of the Indian catfish to identify genes associated with immune function. One thousand nine hundred thirty seven ESTs were submitted to the GenBank with an average read length of approximately 700 bp. Clustering analysis of ESTs yielded 1,698 unique sequences, including 184 contigs and 1,514 singletons. Significant homology to known genes was found by homology searches against data in GenBank in 576 (34 %) ESTs, including similarity to functionally annotated unigenes for 158 ESTs. Additionally, 433 ESTs revealed similarity to unigenes and ESTs in the dbEST but the remaining 658 EST sequences (39 %) did not match any sequence in GenBank. Of a total of 1,698 ESTs generated, 65 ESTs were found to be associated with immune functions. Gene Ontology and KEGG pathway analyses of C. batrachus ESTs collectively revealed a preponderance of immune relevant pathways apart from the presence of pathways involved in protein processing, localization, folding and protein degradation. This study constitutes first EST analysis of lymphoid organ in aquaculturally important Indian catfish species and could pave the way for further research of immune-related genes and functional genomics in this catfish.     

Development of EST derived SSRs and SNPs as a genomic resource in Indian catfish, <Emphasis Type="Italic">Clarias batrachus</Emphasis>

Clarias batrachus, an Indian catfish species, is endemic to the Indian subcontinent and potential cultivable species. The genomic resources in C. batrachus in the form of ESTs containing microsatellite repeats (EST-SSR) and single nucleotide polymorphisms (SNPs) that are associated with the expressed genes from spleen were mined. From a total of 1,937 ESTs generated, 1,698 unique sequences were obtained, out of which 221 EST-SSRs were identified and 54% could be functionally annotated by similarity searches. A total of 23 contigs containing 3 or more ESTs were found to contain 31 SNP loci, out of which 8 ESTs showed similarity to genes of known function and 1 for hypothetical protein. Nine ESTs with SSRs and/or SNPs identified in this study were reported to be associated with diseases in human and animals. These identified loci can be developed into markers in C. batrachus, which can be useful in linkage mapping, comparative genomics studies and for its genetic improvement programmes.     

Use of an expressed sequence tag-based method for single nucleotide polymorphism identification and discrimination of Citrus species and cultivars

Sweet orange [Citrus sinensis (L.) Osbeck] represents the most important Citrus species, followed by clementine (C. clementina Hort. ex Tan.). Citrus species and genotypes are difficult to recognize as they have a moderate level of diversity due to nucellar selection, vegetative propagation and origin by single spontaneous mutation. Despite the large number of available sequences and the existence of a draft assembly of sweet orange and clementine, there are currently no single nucleotide polymorphism (SNP) databases for Citrus species. For this purpose, the QualitySNP software was used to discover SNPs in 19 Citrus species starting from 540,000 expressed sequence tags (ESTs) assembled in 52,000 contigs. The vast majority of ESTs, contigs and SNPs were found in C. clementina and C. sinensis: 4,400 out of 16,000 contigs (27 %) of C. clementina and 4,100 out of 17,000 contigs (24 %) of C. sinensis contained putative SNPs. A total of 3,634 sequences were associated with enzymes belonging to 121 metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, among which the secondary metabolite pathway was the most represented. A total of 163 SNPs from 52 contigs and genes of specific functional categories were validated and 81 polymorphic sites were found. Thirty-seven selected SNPs, validated by Sanger sequencing, confirmed that polymorphisms were mainly between species, while poor within-species variability was discovered. This work provides a collection of 15,879 putative SNP markers that could be exploited by the Citrus community. Furthermore, the validated SNPs associated with specific genes could be used for functional genetic studies in germplasm diversity analysis, mapping and breeding.     

De novo sequencing of hazelnut bacterial artificial chromosomes (BACs) using multiplex Illumina sequencing and targeted marker development for eastern filbert blight resistance

Bacterial artificial chromosome (BAC) libraries are widely used in map-based cloning of plant genes. Eastern filbert blight (EFB), caused by the pyrenomycete Anisogramma anomala (Peck) E. Müller, is a devastating disease of European hazelnut (Corylus avellana L.) in the Pacific Northwest. A dominant allele at a single locus from the obsolete pollenizer “Gasaway” confers complete resistance. Our map-based cloning efforts use a BAC library for “Jefferson” hazelnut, which is heterozygous for resistance. Screening the library with primer pairs designed from RAPD markers closely linked to the EFB resistance locus identified 38 BACs. We sequenced 28 of these BACs using Illumina technology, by multiplexing with barcoded adapters. De novo sequence assembly using the programs Velvet and SOPRA and further alignment using CodonCode Aligner generated contigs whose length ranged from 393 to 108,194 bp. The number of contigs per BAC ranged from 1 to 19, and estimated coverage of assembled BACs ranged from 64 % to 100 %. Preliminary analysis of the sequences identified 779 simple sequence repeats (SSRs), from which we developed 23 markers. Of these, 17 were assigned to linkage group 6 adjacent to the disease resistance locus, five were placed on other linkage groups, and one could not be assigned to a linkage group. The BAC sequences and new SSR markers will be useful for our efforts at map-based cloning of the disease resistance gene.     

Exploiting a wheat EST database to assess genetic diversity

Expressed sequence tag (EST) markers have been used to assess variety and genetic diversity in wheat (Triticum aestivum). In this study, 1549 ESTs from wheat infested with yellow rust were used to examine the genetic diversity of six susceptible and resistant wheat cultivars. The aim of using these cultivars was to improve the competitiveness of public wheat breeding programs through the intensive use of modern, particularly marker-assisted, selection technologies. The F₂ individuals derived from cultivar crosses were screened for resistance to yellow rust at the seedling stage in greenhouses and adult stage in the field to identify DNA markers genetically linked to resistance. Five hundred and sixty ESTs were assembled into 136 contigs and 989 singletons. BlastX search results showed that 39 (29%) contigs and 96 (10%) singletons were homologous to wheat genes. The database-matched contigs and singletons were assigned to eight functional groups related to protein synthesis, photosynthesis, metabolism and energy, stress proteins, transporter proteins, protein breakdown and recycling, cell growth and division and reactive oxygen scavengers. PCR analyses with primers based on the contigs and singletons showed that the most polymorphic functional categories were photosynthesis (contigs) and metabolism and energy (singletons). EST analysis revealed considerable genetic variability among the Turkish wheat cultivars resistant and susceptible to yellow rust disease and allowed calculation of the mean genetic distance between cultivars, with the greatest similarity (0.725) being between Harmankaya99 and Sönmez2001, and the lowest (0.622) between Aytin98 and Izgi01.     

Relationship between homoeologous regulatory and structural genes in allopolyploid genome – A case study in bread wheat

Background

Whole-genome physical maps facilitate genome sequencing, sequence assembly, mapping of candidate genes, and the design of targeted genetic markers. An automated protocol was used to construct a Vitis vinifera 'Cabernet Sauvignon' physical map. The quality of the result was addressed with regard to the effect of high heterozygosity on the accuracy of contig assembly. Its usefulness for the genome-wide mapping of genes for disease resistance, which is an important trait for grapevine, was then assessed.

Results

The physical map included 29,727 BAC clones assembled into 1,770 contigs, spanning 715,684 kbp, and corresponding to 1.5-fold the genome size. Map inflation was due to high heterozygosity, which caused either the separation of allelic BACs in two different contigs, or local mis-assembly in contigs containing BACs from the two haplotypes. Genetic markers anchored 395 contigs or 255,476 kbp to chromosomes. The fully automated assembly and anchorage procedures were validated by BAC-by-BAC blast of the end sequences against the grape genome sequence, unveiling 7.3% of chimerical contigs. The distribution across the physical map of candidate genes for non-host and host resistance, and for defence signalling pathways was then studied. NBS-LRR and RLK genes for host resistance were found in 424 contigs, 133 of them (32%) were assigned to chromosomes, on which they are mostly organised in clusters. Non-host and defence signalling genes were found in 99 contigs dispersed without a discernable pattern across the genome.

Conclusion

Despite some limitations that interfere with the correct assembly of heterozygous clones into contigs, the 'Cabernet Sauvignon' physical map is a useful and reliable intermediary step between a genetic map and the genome sequence. This tool was successfully exploited for a quick mapping of complex families of genes, and it strengthened previous clues of co-localisation of major NBS-LRR clusters and disease resistance loci in grapevine.     

Utility of Host Delivered RNAi of Two FMRF Amide Like Peptides,flp-14 and flp-18, for the Management of Root Knot Nematode,Meloidogyne incognita

Root knot nematode, Meloidogyne incognita, is an obligate sedentary endoparasite that infects a large number of crop species and causes substantial yield losses. Non-chemical based control strategies for these nematodes are gaining importance. In the present study, we have demonstrated the significance of two FMRFamide like peptide genes (flp-14 and flp-18) for infection and development of resistance to M. incognita through host-derived RNAi. The study demonstrated both in vitro and in planta validation of RNAi-induced silencing of the two genes cloned from J2 stage of M. incognita. In vitro silencing of both the genes interfered with nematode migration towards the host roots and subsequent invasion into the roots. Transgenic tobacco lines were developed with RNAi constructs of flp-14 and flp-18 and evaluated against M. incognita. The transformed plants did not show any visible phenotypic variations suggesting the absence of any off-target effects. Bioefficacy studies with deliberate challenging of M. incognita resulted in 50-80% reduction in infection and multiplication confirming the silencing effect. We have provided evidence for in vitro and in planta silencing of the genes by expression analysis using qRT-PCR. Thus the identified genes and the strategy can be used as a potential tool for the control of M. incognita. This is the first ever report that has revealed the utility of host delivered RNAi of flps to control M. incognita. The strategy can also be extended to other crops and nematodes.     

A second-generation integrated map of the silkworm reveals synteny and conserved gene order between lepidopteran insects

A second-generation linkage map was constructed for the silkworm, Bombyx mori, focusing on mapping Bombyx sequences appearing in public nucleotide databases and bacterial artificial chromosome (BAC) contigs. A total of 874 BAC contigs containing 5067 clones (22% of the library) were constructed by PCR-based screening with sequence-tagged sites (STSs) derived from whole-genome shotgun (WGS) sequences. A total of 523 BAC contigs, including 342 independent genes registered in public databases and 85 expressed sequence tags (ESTs), were placed onto the linkage map. We found significant synteny and conserved gene order between B. mori and a nymphalid butterfly, Heliconius melpomene, in four linkage groups (LGs), strongly suggesting that using B. mori as a reference for comparative genomics in Lepidotera is highly feasible.