Chee How Teo

Genome Constitution and Classification Using Retrotransposon- Based Markers in the Orphan Crop Banana

We have exploited the repetitive and dispersed nature of many long terminal repeat (LTR)-retrotransposon families for characterizing genome constitutions and classifying cultivars of the genusMusa. Insertional polymorphisms of the elements were studied using seven published and two newly designed primers facing outwards from the LTRs and reverse transcriptase (RT) domain of the retrotransposon. The primers generated specific amplification patterns showing the universal applicability of this marker type. The Inter-Retrotransposon Amplified Polymorphism (IRAP) markers distinguished the A and B genomes of the banana species (Musa acuminata Col la andMusa balbisiana Colla) and between banana cultivars. The IRAP markers enabled phylogenetic analysis of 16 Malaysian banana cultivars and determination of the genome constitution of hybrid banana (AAB, ABB, AABB, and AAAB), and gave information about ancestral genotypes of the hybrids. In addition, the IRAP detected new retrotransposon insertions into the genome of tissue culture regenerants. This PCR-based IRAP assay is amenable to large-scale throughput demands in screening breeding populations and is applicable for any crop.

Endogenous pararetroviral sequences in tomato (Solanum lycopersicum) and related species

BackgroundEndogenous pararetroviral sequences (EPRVs) are a recently discovered class of repetitive sequences that is broadly distributed in the plant kingdom. The potential contribution of EPRVs to plant pathogenicity or, conversely, to virus resistance is just beginning to be explored. Some members of the family Solanaceae are particularly rich in EPRVs. In previous work, EPRVs have been characterized molecularly in various species of Nicotiana including N.tabacum (tobacco) and Solanum tuberosum (potato). Here we describe a family of EPRVs in cultivated tomato (Solanum lycopersicum L.) and a wild relative (S.habrochaites).ResultsMolecular cloning and DNA sequence analysis revealed that tomato EPRVs (named LycEPRVs) are most closely related to those in tobacco. The sequence similarity of LycEPRVs in S.lycopersicum and S.habrochaites indicates they are potentially derived from the same pararetrovirus. DNA blot analysis revealed a similar genomic organization in the two species, but also some independent excision or insertion events after species separation, or flanking sequence divergence. LycEPRVs share with the tobacco elements a disrupted genomic structure and frequent association with retrotransposons. Fluorescence in situ hybridization revealed that copies of LycEPRV are dispersed on all chromosomes in predominantly heterochromatic regions. Methylation of LycEPRVs was detected in CHG and asymmetric CHH nucleotide groups. Although normally quiescent EPRVs can be reactivated and produce symptoms of infection in some Nicotiana interspecific hybrids, a similar pathogenicity of LycEPRVs could not be demonstrated in Solanum L. section Lycopersicon [Mill.] hybrids. Even in healthy plants, however, transcripts derived from multiple LycEPRV loci and short RNAs complementary to LycEPRVs were detected and were elevated upon infection with heterologous viruses encoding suppressors of PTGS.ConclusionThe analysis of LycEPRVs provides further evidence for the extensive invasion of pararetroviral sequences into the genomes of solanaceous plants. The detection of asymmetric CHH methylation and short RNAs, which are hallmarks of RNAi in plants, suggests that LycEPRVs are controlled by an RNA-mediated silencing mechanism.

Telomere-mediated truncation of barley chromosomes

Engineered minichromosomes offer an enormous opportunity to plant biotechnology as they have the potential to simultaneously transfer and stably express multiple genes. Following a top-down approach, we truncated endogenous chromosomes in barley (Hordeum vulgare) by Agrobacterium-mediated transfer of T-DNA constructs containing telomere sequences. Blocks of Arabidopsis-like telomeric repeats were inserted into a binary vector suitable for stable transformation. After transfer of these constructs into immature embryos of diploid and tetraploid barley, chromosome truncation by T-DNA-induced de novo formation of telomeres could be confirmed by fluorescent in situ hybridisation, primer extension telomere repeat amplification and DNA gel blot analysis in regenerated plants. Telomere seeding connected to chromosome truncation was found in tetraploid plants only, indicating that genetic redundancy facilitates recovery of shortened chromosomes. Truncated chromosomes were transmissible in sexual reproduction, but were inherited at rates lower than expected according to Mendelian rules.

Evolutionary dynamics and sites of illegitimate recombination revealed in the interspersion and sequence junctions of two nonhomologous satellite DNAs in cactophilic Drosophila species

Satellite DNA (satDNA) is a major component of genomes but relatively little is known about the fine-scale organization of unrelated satDNAs residing at the same chromosome location, and the sequence structure and dynamics of satDNA junctions. We studied the organization and sequence junctions of two nonhomologous satDNAs, pBuM and DBC-150, in three species from the neotropical Drosophila buzzatii cluster (repleta group). In situ hybridization to microchromosomes, interphase nuclei and extended DNA fibers showed frequent interspersion of the two satellites in D. gouveai, D. antonietae and, to a lesser extent, D. seriema. We isolated by PCR six pBuM × DBC-150 junctions: four are exclusive to D. gouveai and two are exclusive to D. antonietae. The six junction breakpoints occur at different positions within monomers, suggesting independent origin. Four junctions showed abrupt transitions between the two satellites, whereas two junctions showed a distinct 10 bp tandem duplication before the junction. Unlike pBuM, DBC-150 junction repeats are more variable than randomly cloned monomers and showed diagnostic features in common to a 3-monomer higher-order repeat seen in the sister species D. serido. The high levels of interspersion between pBuM and DBC-150 repeats suggest extensive rearrangements between the two satellites, maybe favored by specific features of the microchromosomes. Our interpretation is that the junctions evolved by multiples events of illegitimate recombination between nonhomologous satDNA repeats, with subsequent rounds of unequal crossing-over expanding the copy number of some of the junctions.

The cloning of Ty1-copia-like retrotransposons from 10 varieties of banana (Musa Sp.).

Ty1-copia-like retrotransposons have been identified and investigated in several plant species. Here, the internal region of the reverse transcriptase (RT) gene of Ty1-copia-like retrotransposons was amplified by PCR from total genomic DNA of 10 varieties of banana. Two to four clones from each variety were sequenced. Extreme heterogeneity in the sequences of Ty1-copia-like retrotransposons from all the varieties was revealed following sequence analysis of the reverse transcriptase (RT) fragments. The size of the individual RT gene fragments varied between 213 and 309 bp. Southern blots of genomic DNA digested from Musa acuminata and other banana varieties probed with W8 clone from M. acuminata and A4 clone from Pisang Abu Nipah showed similar strong, multiple restriction fragments together with other faint hybridization band patterns with variable intensities indicating the presence of many copies of the Ty1-copia-like retrotransposons in the genomes. There was no correlation between retroelement sequence and the banana species (with A or B genomes) from which it arose, suggesting that the probes are not useful for tracking genomes through breeding populations.

De novo generation of plant centromeres at tandem repeats

Artificial minichromosomes are highly desirable tools for basic research, breeding, and biotechnology purposes. We present an option to generate plant artificial minichromosomes via de novo engineering of plant centromeres in Arabidopsis thaliana by targeting kinetochore proteins to tandem repeat arrays at non-centromeric positions. We employed the bacterial lactose repressor/lactose operator system to guide derivatives of the centromeric histone H3 variant cenH3 to LacO operator sequences. Tethering of cenH3 to non-centromeric loci led to de novo assembly of kinetochore proteins and to dicentric carrier chromosomes which potentially form anaphase bridges. This approach will be further developed and may contribute to generating minichromosomes from preselected genomic regions, potentially even in a diploid background.

Engineered plant minichromosomes

Minichromosomes offer an enormous potential for plant breeding and biotechnology, because they may simultaneously transfer and stably express multiple genes. Segregating independently of their host chromosomes, they provide a platform for accelerating plant breeding. Minichromosomes can be established from cloned components in vivo (bottom up) or via engineering of natural chromosomes (top down). When they possess functional centromeres and telomeres, they should be stably inherited, but their meiotic transmission rate is below that of endogenous chromosomes. To achieve the customized generation and control the regular transmission of minichromosomes are important challenges for applied research in chromosome biology. Here, construction and biology of plant minichromosomes are compared with data available for yeast and animal systems.

Comparative analysis of Argonaute gene sequences in bananas (Musa sp.) shows conserved species-specific Ago-7 PIWI domains

The Argonaute proteins are key components in the effector complex of RNA silencing pathways which interact with small RNAs to mediate sequence specific silencing of nucleic acid targets. In plants, these proteins are involved in a diversity of biological roles such as antiviral defence, heterochromatin regulation and in the regulation of growth and development. This report describes the study of Argonaute gene sequences in bananas which are an important food staple for many developing nations. This study successfully isolated AGO7-specific PIWI domain genomic sequences from 12 diploid Musa species of three Musa sections and also from Ensete. The Musa AGO7-specific PIWI domain sequences showed the highest similarity to rice AGO7/SHOOTLESS4 with an average amino acid identity of 78%. Phylogenetic analysis of the Musa sequences revealed phylogenetic grouping that agrees fairly well with the present knowledge of the taxonomic classification of Musa species. In addition, this study estimated that there are at least 15 Argonaute genes or loci containing PIWI domain sequences in the genome of Musa acuminata ssp. malaccensis.

An improved method for inducing prometaphase chromosomes in plants

BackgroundDetailed karyotyping using metaphase chromosomes in melon (Cucumis melo L.) remains a challenge because of their small chromosome sizes and poor stainability. Prometaphase chromosomes, which are two times longer and loosely condensed, provide a significantly better resolution for fluorescence in situ hybridization (FISH) than metaphase chromosomes. However, suitable method for acquiring prometaphase chromosomes in melon have been poorly investigated.ResultsIn this study, a modified Carnoy’s solution II (MC II) [6:3:1 (v/v) ethanol: acetic acid: chloroform] was used as a pretreatment solution to obtain prometaphase chromosomes. We demonstrated that the prometaphase chromosomes obtained using the MC II method are excellent for karyotyping and FISH analysis. We also observed that a combination of MC II and the modified air dry (ADI) method provides a satisfactory meiotic pachytene chromosome preparation with reduced cytoplasmic background and clear chromatin spreads. Moreover, we demonstrated that pachytene and prometaphase chromosomes of melon and Abelia × grandiflora generate significantly better FISH images when prepared using the method described. We confirmed, for the first time, that Abelia × grandiflora has pairs of both strong and weak 45S ribosomal DNA signals on the short arms of their metaphase chromosomes.ConclusionThe MC II and ADI method are simple and effective for acquiring prometaphase and pachytene chromosomes with reduced cytoplasm background in plants. Our methods provide high-resolution FISH images that can help accelerate molecular cytogenetic research in plants.