Bacterial artificial chromosome (BAC) library resource for positional cloning of pest and disease resistance genes in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Pest and disease problems are important constraints of cassava production and host plant resistance is the most efficient method of combating them. Breeding for host plant resistance is considerably slowed down by the crop’s biological constraints of a long growth cycle, high levels of heterozygosity and a large genetic load. More efficient methods such as gene cloning and transgenesis are required to deploy resistance genes. To facilitate the cloning of resistance genes, bacterial artificial chromosome (BAC) library resources have been developed for cassava. Two libraries were constructed from the cassava clones, TMS 30001, resistant to the cassava mosaic disease (CMD) and the cassava bacterial blight (CBB), and MECU72, resistant to cassava white fly. The TMS30001 library has 55 296 clones with an insert size range of 40–150 kb with an average of 80 kb, while the MECU72 library consists of 92 160 clones and an insert size range of 25–250 kb average of 93 kb. Based on a genome size of 772 Mb, the TMS30001 and MECU72 libraries have a 5 and 11.3 haploid genome equivalents and a 95 and 99 chance of finding any sequence, respectively. To demonstrate the potential of the libraries, the TMS30001 library was screened by southern hybridization using a cassava analog (CBB1) of the Xa21 gene from rice that maps to a region containing a QTL for resistance to CBB as probe. Five BAC clones that hybridized to CBB1 were isolated and a Hind III fingerprint revealed 2–3 copies of the gene in individual BAC clones. A larger scale analysis of resistance gene analogs (RGAs) in cassava has also been conducted in order to understand the number and organization of RGAs. To scan for gene and repeat DNA content in the libraries, end-sequencing was performed on 2301 clones from the MECU72 library. A total of 1705 unique sequences were obtained with an average size of 715 bp. Database homology searches using BLAST revealed that 458 sequences had significant homology with known proteins and 321 with transposable elements. The use of the library in positional cloning of pest and disease resistance genes is discussed.     

Isolation of Resistance Gene Candidates (RGCs) and characterization of an RGC cluster in cassava

Plant disease resistance genes (R genes) show significant similarity amongst themselves in terms of both their DNA sequences and structural motifs present in their protein products. Oligonucleotide primers designed from NBS (Nucleotide Binding Site) domains encoded by several R-genes have been used to amplify NBS sequences from the genomic DNA of various plant species, which have been called Resistance Gene Analogues (RGAs) or Resistance Gene Candidates (RGCs). Using specific primers from the NBS and TIR (Toll/Interleukin-1 Receptor) regions, we identified twelve classes of RGCs in cassava ( Manihot esculenta Crantz). Two classes were obtained from the PCR-amplification of the TIR domain. The other 10 classes correspond to the NBS sequences and were grouped into two subfamilies. Classes RCa1 to RCa5 are part of the first subfamily and were linked to a TIR domain in the N terminus. Classes RCa6 to RCa10 corresponded to non-TIR NBS-LRR encoding sequences. BAC library screening with the 12 RGC classes as probes allowed the identification of 42 BAC clones that were assembled into 10 contigs and 19 singletons. Members of the two TIR and non-TIR NBS-LRR subfamilies occurred together within individual BAC clones. The BAC screening and Southern hybridization analyses showed that all RGCs were single copy sequences except RCa6 that represented a large and diverse gene family. One BAC contained five NBS sequences and sequence analysis allowed the identification of two complete RGCs encoding two highly similar proteins. This BAC was located on linkage group J with three other RGC-containing BACs. At least one of these genes, RGC2, is expressed constitutively in cassava tissues.Communicated by M.-A. Grandbastien     

Gene expression profile in response to <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">manihotis</Emphasis> infection in cassava using a cDNA microarray

A cassava cDNA microarray based on a large cassava EST database was constructed and used to study the incompatible interaction between cassava and Xanthomonas axonopodis pv. manihotis (Xam) strain CIO151. For microarray construction, 5700 clones from the cassava unigene set were amplified by polymerase chain reaction (PCR) and printed on glass slides. Microarray hybridization was performed using cDNA from cassava plants (resistant variety MBra685) collected at 12, 24, 48 h and 7 and 15 days post-infection as treatment and cDNA from mock-inoculated plants as control. A total of 199 genes were found to be differentially expressed (126 up-regulated and 73 down-regulated). A greater proportion of differentially-expressed genes was observed at 7 days after inoculation. Expression profiling and cluster analyses indicate that, in response to inoculation with Xam, cassava induces dozens of genes, including principally those involved in oxidative burst, protein degradation and pathogenesis-related (PR) genes. In contrast, genes encoding proteins that are involved in photosynthesis and metabolism were down regulated. In addition, various other genes encoding proteins with unknown function or showing no similarity to other proteins were also induced. Quantitative real time PCR experiments confirmed the reliability of our microarray data. In addition we showed that some genes are induced more rapidly in the resistant than in the susceptible cultivar.These authors made equal contributions to this work.     

Differential expression of miRNAs and their target genes in senescing leaves and siliques: insights from deep sequencing of small RNAs and cleaved target RNAs

MicroRNAs (miRNAs) are a class of small RNAs, which typically function by guiding cleavage of target mRNAs. They are known to play roles in a variety of plant processes including development, responses to environmental stresses and senescence. To identify senescence regulation of miRNAs in Arabidopsis thaliana, eight small RNA libraries were constructed and sequenced at four different stages of development and senescence from both leaves and siliques, resulting in more than 200 million genome‐matched sequences. Parallel analysis of RNA ends libraries, which enable the large‐scale examination of miRNA‐guided cleavage products, were constructed and sequenced, resulting in over 750 million genome‐matched sequences. These large datasets led to the identification a new senescence‐inducible small RNA locus, as well as new regulation of known miRNAs and their target genes during senescence, many of which have established roles in nutrient responsiveness and cell structural integrity. In keeping with remobilization of nutrients thought to occur during senescence, many miRNAs and targets had opposite expression pattern changes between leaf and silique tissues during the progression of senescence. Taken together, these findings highlight the integral role that miRNAs may play in the remobilization of resources and alteration of cellular structure that is known to occur in senescence.     

The α-gliadin gene family. I. Characterization of ten new wheat α-gliadin genomic clones, evidence for limited sequence conservation of flanking DNA, and Southern analysis of the gene family

 A detailed study of the organization of the wheat α-gliadin gene family is described. In the first stage of this study recombinant libraries of sufficient size and quality were constructed to allow examination of large, closely-related, gene families. A large number of α-gliadin clones were then isolated in a partial screen of these libraries. A set of these clones were sequenced and ten new gene sequences are now reported. Including these new sequences, 20 of the 27 known α-gliadin genomic and cDNA sequences are from cv Cheyenne, making this cultivar the best studied for gliadin family organization. An analysis of the DNA flanking the coding regions shows divergences which may indicate the functional ends of the α-gliadin genes. High-resolution Southern analyses on DNA from aneuploid and chromosome-substitution lines allow most of the 20 distinct α-gliadin HindIII restriction fragments in cvs Cheyenne and Chinese Spring to be assigned to specific genomes. It is estimated that as many as 150 α-gliadin genes occur in the cultivar Cheyenne, and the limitations of such estimates are discussed. Received: 1 October 1996 / Accepted: 20 December 1996     

EST-based gene discovery in pig: virtual expression patterns and comparative mapping to human

A molecular understanding of porcine reproduction is of biological interest and economic importance. Our Midwest Consortium has produced cDNA libraries containing the majority of genes expressed in major female reproductive tissues, and we have deposited into public databases 21,499 expressed sequence tag (EST) gene sequences from the 3 end of clones from these libraries. These sequences represent 10,574 different genes, based on sequence comparison among these data, and comparison with existing porcine ESTs and genes indicate as many as 4652 of these EST clusters are novel. In silico analysis identified sequences that are expressed in specific pig tissues or organs and confirmed the broad expression in pig for many genes ubiquitously expressed in human tissues. Furthermore, we have developed computer software to identify sequence similarity of these pig genes with their human counterparts, and to extract the mapping information of these human homologues from genome databases. We demonstrate the utility of this software for comparative mapping by localizing 61 genes on the porcine physical map for Chromosomes (Chrs) 5, 10, and 14. The following Accession numbers were assigned to our deposited sequences: BF701840 – BF704551, BF708383, BF708386 – BF713604, BG322266 – BG322271, BI398567 – BI405235, BQ597354 – BQ605166.     

An ordered EST catalogue and gene expression profiles of cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis>) at key growth stages

A cDNA library was constructed from the root tissues of cassava variety Huanan 124 at the root bulking stage. A total of 9,600 cDNA clones from the library were sequenced with single-pass from the 5′-terminus to establish a catalogue of expressed sequence tags (ESTs). Assembly of the resulting EST sequences resulted in 2,878 putative unigenes. Blastn analysis showed that 62.6% of the unigenes matched with known cassava ESTs and the rest had no ‘hits’ against the cassava database in the integrative PlantGDB database. Blastx analysis showed that 1,715 (59.59%) of the unigenes matched with one or more GenBank protein entries and 1,163 (40.41%) had no ‘hits’. A cDNA microarray with 2,878 unigenes was developed and used to analyze gene expression profiling of Huanan 124 at key growth stages including seedling, formation of root system, root bulking, and starch maturity. Array data analysis revealed that (1) the higher ratio of up-regulated ribosome-related genes was accompanied by a high ratio of up-regulated ubiquitin, proteasome-related and protease genes in cassava roots; (2) starch formation and degradation simultaneously occur at the early stages of root development but starch degradation is declined partially due to decrease in UDP-glucose dehydrogenase activity with root maturity; (3) starch may also be synthesized in situ in roots; (4) starch synthesis, translocation, and accumulation are also associated probably with signaling pathways that parallel Wnt, LAM, TCS and ErbB signaling pathways in animals; (5) constitutive expression of stress-responsive genes may be due to the adaptation of cassava to harsh environments during long-term evolution.     

Construction of two BAC libraries from the wild Mexican diploid potato,<Emphasis Type="Italic"> Solanum pinnatisectum,</Emphasis> and the identification of clones near the late blight and Colorado potato beetle resistance loci

To facilitate isolation and characterization of disease and insect resistance genes important to potato, two bacterial artificial chromosome (BAC) libraries were constructed from genomic DNA of the Mexican wild diploid species, Solanum pinnatisectum, which carries high levels of resistance to the most important potato pathogen and pest, the late blight and the Colorado potato beetle (CPB). One of the libraries was constructed from the DNA, partially digested with BamHI, and it consists of 40,328 clones with an average insert size of 125 kb. The other library was constructed from the DNA partially digested with EcoRI, and it consists of 17,280 clones with an average insert size of 135 kb. The two libraries, together, represent approximately six equivalents of the wild potato haploid genome. Both libraries were evaluated for contamination with organellar DNA sequences and were shown to have a very low percentage (0.65–0.91%) of clones derived from the chloroplast genome. High-density filters, prepared from the two libraries, were screened with ten restriction fragment length polymorphism (RFLP) markers linked to the resistance genes for late blight, CPB, Verticillium wilt and potato cyst nematodes, and the gene Sr1 for the self-incompatibility S-locus. Thirty nine positive clones were identified and at least two positive BAC clones were detected for each RFLP marker. Four markers that are linked to the late blight resistance gene Rpi1 hybridized to 14 BAC clones. Fifteen BAC clones were shown to harbor the PPO (polyphenol oxidase) locus for the CPB resistance by three RFLP probes. Two RFLP markers detected five BAC clones that were linked to the Sr1 gene for self-incompatibility. These results agree with the librarys predicted extent of coverage of the potato genome, and indicated that the libraries are useful resources for the molecular isolation of disease and insect resistance genes, as well as other economically important genes in the wild potato species. The development of the two potato BAC libraries provides a starting point, and landmarks for BAC contig construction and chromosome walking towards the map-based cloning of agronomically important target genes in the species.Communicated by H.F. Linskens     

A geminivirus-induced gene silencing system for gene function validation in cassava

We have constructed an African cassava mosaic virus (ACMV) based gene-silencing vector as a reverse genetics tool for gene function analysis in cassava. The vector carrying a fragment from the Nicotiana tabacumsulfur gene (su), encoding one unit of the chloroplast enzyme magnesium chelatase, was used to induce the silencing of the cassava orthologous gene resulting in yellow–white spots characteristic of the inhibition of su expression. This result suggests that well developed sequence databases from model plants like Arabidopsis thaliana, Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum and others could be used as a major source of information and sequences for functional genomics in cassava. Furthermore, a fragment of the cassava CYP79D2endogenous gene, sharing 89% homology with CYP79D1endogenous gene was inserted into the ACMV vector. The resultant vector was inducing the down regulation of the expression of these two genes which catalyze the first-dedicated step in the synthesis of linamarin, the major cyanogenic glycoside in cassava. At 21 days post-inoculation (dpi), a 76% reduction of linamarin content was observed in silenced leaves. Using transgenic plants expressing antisense RNA of CYP79D1and CYP79D2, Siritunga and Sayre (2003) obtained several lines with a reduction level varying from 60% to 94%. This result provides the first example of direct comparison of the efficiency of a virus-induced gene silencing (VIGS) system and the transgenic approach for suppression of a biosynthetic pathway. The ACMV VIGS system will certainly be a complement and in some cases an alternative to the transgenic approach, for gene discovery and gene function analysis in cassava.     

The <Emphasis Type="Italic">NOLA2</Emphasis> and <Emphasis Type="Italic">RPS3A</Emphasis> genes as highly informative markers of human squamous cell carcinoma of lung

cDNA libraries enriched with sequences that are differentially transcribed in normal and tumor tissues were prepared using the subtractive hybridization of mixtures of cDNAs from ten patients with squamous cell carcinoma (lung cancer) and the corresponding mixtures of cDNAs from normal tissues of the same patients. An analysis of the libraries revealed two genes, NOLA2 and RPS3A, whose expression in patients with squamous cell carcinoma increased by 70%. A high frequency of enhanced expression of these genes in the cancer makes them highly informative markers of squamous cell carcinoma, which, together with other markers, can be used for reliable diagnosis of the disease.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 195–199.Original Russian Text Copyright © 2005 by Slizhikova, Vinogradova, Sverdlov.     

Tissue expression map of a large number of expressed sequence tags and its application to in silico screening of stress response genes in common wheat

In order to assess global changes in gene expression patterns in stress-induced tissues, we conducted large-scale analysis of expressed sequence tags (ESTs) in common wheat. Twenty-one cDNA libraries derived from stress-induced tissues, such as callus, as well as liquid cultures and abiotic stress conditions (temperature treatment, desiccation, photoperiod, moisture and ABA) were constructed. Several thousand colonies were randomly selected from each of these 21 cDNA libraries and sequenced from both the 5′ and 3′ ends. By computing abundantly expressed ESTs, correlated expression patterns of genes across the tissues were monitored. Furthermore, the relationships between gene expression profiles among the stress-induced tissues were inferred from the gene expression patterns. Multi-dimensional analysis of EST data is analogous to microarray experiments. As an example, genes specifically induced and/or suppressed by cold acclimation and heat-shock treatments were selected in silico. Four hundred and ninety genes showing fivefold induction or 218 genes for suppression in comparison to the control expression level were selected. These selected genes were annotated with the BLAST search. Furthermore, gene ontology was conducted for these genes with the InterPro search. Because genes regulated in response to temperature treatment were successfully selected, this method can be applied to other stress-treated tissues. Then, the method was applied to screen genes in response to abiotic stresses such as drought and ABA treatments. In silico selection of screened genes from virtual display should provide a powerful tool for functional plant genomics.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Establishment of a high throughput EST sequencing system using poly(A) tail-removed cDNA libraries and determination of 36 000 bovine ESTs

We determined 36 310 bovine expressed sequence tag (EST) sequences using 10 different cDNA libraries. For massive EST sequencing, we devised a new system with two major features. First, we constructed cDNA libraries in which the poly(A) tails were removed using nested deletion at the 3′-ends. This permitted high quality reading of sequences from the 3′-end of the cDNA, which is otherwise difficult to do. Second, we increased throughput by sequencing directly on templates generated by colony PCR. Using this system, we determined 600 cDNA sequences per day. The read-out length was >450 bases in >90% of the sequences. Furthermore, we established a data management system for analyses, storage and manipulation of the sequence data. Finally, 16 358 non-redundant ESTs were derived from ~6900 independent genes. These data will facilitate construction of a precise comparative map across mammalian species and isolate the functional genes that govern economic traits. This system is applicable to other organisms, including livestock, for which EST data are limited.     

An EST survey of the sugarcane transcriptome

Its large genome and high polyploidy makes sugarcane (Saccharum spp.) a singularly challenging crop to study and improve using genetic approaches. To provide large numbers of functionally characterized candidate genes that might be tested for direct association (rather than distant linkage) with economically important traits, we sequenced the 5' ends of 9,216 clones from three cDNA libraries (apex, leaf and mature internode), representing 3,401 non-redundant sequences. About 57% of these sequences could be assigned a tentative function based on statistically significant similarity to previously characterized proteins or DNA sequences. Another 28% corresponded to previously identified, but uncharacterized, sequences. Some of the remaining unidentified sequences were predicted to be genes which could potentially be new to plants or unique to sugarcane. Comparisons of the sugarcane ESTs to a large sorghum EST database revealed similar compositions of expressed genes between some different tissues. Comparison to a detailed Arabidopsis protein database showed some highly conserved sequences, which might be useful DNA markers for pan-angiosperm comparative mapping. These EST sequences provide a foundation for many new studies to accelerate isolation of agronomically important genes from the cumbersome sugarcane genome.