Histology of somatic embryo initiation and organogenesis from rhizome explants of Musa spp.

The origin of somatic embryos derived from rhizome explants of triploid Musa cv. Grand Nain was the subject of histological studies during different phases of ontogenetic development. The investigation revealed that the majority of somatic embryos showed normal root formation and consisted of highly vacuolated cells in the poorly structured shoot apex. The embryogenic mass and somatic embryos were mostly derived from several morphologically competent cells. Single cell origin depended on the presence of organogenetically functional vascular cells of rhizome explants and occurred infrequently. The implications of these findings for genetic improvement of banana and plantain by in vitro mutation breeding and gene technology are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Structural organization of the barley D-hordein locus in comparison with its orthologous regions of wheat genomes.

D hordein, a prolamin storage protein of barley endosperms, is highly homologous to the high molecular weight (HWM) glutenin subunits, which are the major determinants of bread-making quality in wheat flour. In hexaploid wheat (AABBDD), each genome contains two paralogous copies of HMW-glutenin genes that encode the x- and y-type HMW-glutenin subunits. Previously, we reported the sequence analysis of a 102-kb genomic region that contains the HMW-glutenin locus of the D genome from Aegilops tauschii, the donor of the D genome of hexaploid wheat. Here, we present the sequence analysis of a 120-kb D-hordein region of the barley genome, a more distantly related member of the Triticeae grass tribe. Comparative sequence analysis revealed that gene content and order are generally conserved. Genes included in both of these orthologous regions are arranged in the following order: a Xa21-like receptor kinase, an endosperm globulin, an HMW prolamin, and a serine (threonine) protein kinase. However, in the wheat D genome, a region containing both the globulin and HMW-glutenin gene was duplicated, indicating that this duplication event occurred after the separation of the wheat and barley genomes. The intergenic regions are divergent with regard to the sequence and structural organization. It was found that different types of retroelements are responsible for the intergenic structure divergence in the wheat and barley genomes. In the barley region, we identified 16 long terminal repeat (LTR) retrotransposons in three distinct nested clusters. These retroelements account for 63% of the contig sequence. In addition, barley D hordein was compared with wheat HMW glutenins in terms of cysteine residue conservation and repeat domain organization.     

A single Banana streak virus integration event in the banana genome as the origin of infectious endogenous pararetrovirus

Sequencing of plant nuclear genomes reveals the widespread presence of integrated viral sequences known as endogenous pararetroviruses (EPRVs). Banana is one of the three plant species known to harbor infectious EPRVs. Musa balbisiana carries integrated copies of Banana streak virus (BSV), which are infectious by releasing virions in interspecific hybrids. Here, we analyze the organization of the EPRV of BSV Goldfinger (BSGfV) present in the wild diploid M. balbisiana cv. Pisang Klutuk Wulung (PKW) revealed by the study of Musa bacterial artificial chromosome resources and interspecific genetic cross. cv. PKW contains two similar EPRVs of BSGfV. Genotyping of these integrants and studies of their segregation pattern show an allelic insertion. Despite the fact that integrated BSGfV has undergone extensive rearrangement, both EPRVs contain the full-length viral genome. The high degree of sequence conservation between the integrated and episomal form of the virus indicates a recent integration event; however, only one allele is infectious. Analysis of BSGfV EPRV segregation among an F1 population from an interspecific genetic cross revealed that these EPRV sequences correspond to two alleles originating from a single integration event. We describe here for the first time the full genomic and genetic organization of the two EPRVs of BSGfV present in cv. PKW in response to the challenge facing both scientists and breeders to identify and generate genetic resources free from BSV. We discuss the consequences of this unique host-pathogen interaction in terms of genetic and genomic plant defenses versus strategies of infectious BSGfV EPRVs.     

A new database on polymorphic retroelements in human genome (PRED)

Comparison of primate genomes sequences has confirmed the evidence that substantial part of intra- and interspecies differences is provided by retroelements. Human genome contains thousands of polymorphic retroelement copies considered to be perspective molecular genetic markers of new generation. However utilization of polymorphic retroelements as molecular genetic markers is limited due to lack of systematic data on their number, genomic context and distribution among human populations. We have created first bilingual (Russian/English) internet-resource devoted to known polymorphic retroelements discovered in human genome by our group as well as by other researchers worldwide. The database contains information about each retroelement copy location, position relative to known and predicted genes, frequency of alleles in human populations and others. Our internet portal allows to perform a search in database using multiple search conditions and available on http://labcfg.ibch.ru/home.html. The database provides an opportunity to investigate distribution of polymorphic retroelements in human genome and to design new genetic markers for various population and medical studies.     

A homologous protein-coding sequence in Drosophila homeotic genes and its conservation in other metazoans

The homeotic genes of the bithorax complex (BX-C) and the Antennapedia complex (ANT-C) of Drosophila appear to specify the developmental fate of segments or parts of segments of the fly. We have previously reported weak DNA sequence homology between 3' portions of the Antennapedia and fushi tarazu genes of the ANT-C and the Ultrabithorax gene of the BX-C. Here we show that this DNA homology (the homeo box) is due to a conserved protein-coding sequence present in these three pattern-formation genes. Thus the functional homology between these developmental controlling genes is reflected in a structural homology in their gene products. The homeo box sequence is also present in a few copies in the genomes of some other invertebrates, and is even conserved in vertebrate genomes, including the human genome. Apparently at least a part of these developmental switch genes from Drosophila is highly conserved during evolution, and might perform an analogous function in many metazoans .     

Phylogenetic and molecular analysis of the ribulose-1,5-bisphosphate carboxylase small subunit gene family in banana

Despite being the number one fruit crop in the world, very little is known about the phylogeny and molecular biology of banana (Musa spp.). Six banana rbcS gene families encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from six different Musa spp. are presented. For a comprehensive phylogenetic study using Musa rbcS genes, a total of 57 distinct rbcS sequences was isolated from six accessions that contained different combinations of the A and B ancestral/parental genomes. As a result, five of the six members of the rbcS gene family could be affiliated with the A and/or B Musa genomes and at least three of the six gene families most likely existed before Musa A and B genomes separated. By combining sequence data with quantitative real-time PCR it was determined that the different Musa rbcS gene family members are also often multiply represented in each genome, with the highest copy numbers in the B genome. Expression of some of the rbcS genes varied in intensity and in different tissues indicating differences in regulation. To analyse and compare regulatory sequences of Musa rbcS genes, promoter and terminator regions were cloned for three Musa rbcS genes. Transient transformation assays using promoter-reporter-terminator constructs in maize, wheat, and sugarcane demonstrated that the rbcS-Ma1, rbcS-Ma3, and rbcS-Ma5 promoters could be useful for transgene expression in heterologous expression systems. Furthermore, the rbcS-Ma1 terminator resulted in a 2-fold increase of transgene expression when directly compared with the widely used Nos terminator.     

MAGGY, a retrotransposon in the genome of the rice blast fungusMagnaporthe grisea

Full-length copies of a previously described repetitive DNA sequence (CH2-8) were isolated from the genome of theMagnaporthe grisea strain 2539. One copy of the complete element was sequenced and found to resemble agypsy-like LTR retrotransposon. We named this element MAGGY (MAGnaporthe GYpsy-like element). MAGGY contains two internal ORFs putatively encoding Gag, Pol and Env-like proteins which are similar to peptides encoded by retroelements identified in other filamentous fungi. MAGGY was found to be widely distributed amongM. grisea isolates from geographically dispersed locations and different hosts. It was present in high copy number in the genomes of all nine rice-pathogenic isolates examined. By contrast,M. grisea strains isolated from other Gramineae were found to possess varying copy numbers of MAGGY and in some cases the element was completely absent. The wide distribution of MAGGY suggests that this element invaded the genome ofM. grisea prior to the evolution of rice-specific form(s). It may since have been horizontally transmitted to other sub-specific groups. One copy of MAGGY, corresponding to the element we sequenced, was located at identical locations in the genomes of geographically dispersed strains, suggesting that this copy of the element is a relatively ancient insertion.     

MAGGY,a retrotransposon in the genome of the rice blast fungusMagnaporthe grisea

Full-length copies of a previously described repetitive DNA sequence (CH2-8) were isolated from the genome of theMagnaporthe grisea strain 2539. One copy of the complete element was sequenced and found to resemble agypsy-like LTR retrotransposon. We named this element MAGGY (MAGnaporthe GYpsy-like element). MAGGY contains two internal ORFs putatively encoding Gag, Pol and Env-like proteins which are similar to peptides encoded by retroelements identified in other filamentous fungi. MAGGY was found to be widely distributed amongM. grisea isolates from geographically dispersed locations and different hosts. It was present in high copy number in the genomes of all nine rice-pathogenic isolates examined. By contrast,M. grisea strains isolated from other Gramineae were found to possess varying copy numbers of MAGGY and in some cases the element was completely absent. The wide distribution of MAGGY suggests that this element invaded the genome ofM. grisea prior to the evolution of rice-specific form(s). It may since have been horizontally transmitted to other sub-specific groups. One copy of MAGGY, corresponding to the element we sequenced, was located at identical locations in the genomes of geographically dispersed strains, suggesting that this copy of the element is a relatively ancient insertion.     

Genome-Wide Comparative Analysis of pogo-Like Transposable Elements in Different Fusarium Species

The recent availability of genome sequences of four different Fusarium species offers the opportunity to perform extensive comparative analyses, in particular of repeated sequences. In a recent work, the overall content of such sequences in the genomes of three phylogenetically related Fusarium species, F. graminearum, F. verticillioides, and F. oxysporum f. sp. lycopersici has been estimated. In this study, we present an exhaustive characterization of pogo-like elements, named Fots, in four Fusarium genomes. Overall 10 Fot and two Fot-related miniature inverted-repeat transposable element families were identified, revealing a diversification of multiple lineages of pogo-like elements, some of which accompanied by a gain of introns. This analysis also showed that such elements are present in an unusual high proportion in the genomes of F. oxysporum f. sp. lycopersici and Nectria haematococca (anamorph F. solani f. sp. pisi) in contrast with most other fungal genomes in which retroelements are the most represented. Interestingly, our analysis showed that the most numerous Fot families all contain potentially active or mobilisable copies, thus conferring a mutagenic potential of these transposable elements and consequently a role in strain adaptation and genome evolution. This role is strongly reinforced when examining their genomic distribution which is clearly biased with a high proportion (more than 80%) located on strain- or species-specific regions enriched in genes involved in pathogenicity and/or adaptation. Finally, the different reproductive characteristics of the four Fusarium species allowed us to investigate the impact of the process of repeat-induced point mutations on the expansion and diversification of Fot elements.     

MITE类转座子mPing在水稻不同亚种间的差异分析

mPing是水稻中第一个被鉴定出的有活性的MITE类转座子,为了探索mPing在水稻粳稻品种日本晴和籼稻品种93-11基因组中的分布差异,本研究首先运用Southern杂交的方法初步检测m Ping在两个亚种中拷贝数的差异,然后通过同源性探寻方法发现,m Ping在水稻亚种日本晴和93-11基因组中拷贝数分别为52和14,并且日本晴基因组中的m Ping均为m Ping-1,93-11中m Ping-1的拷贝数为3,m Ping-2的拷贝数为11。通过分析m Ping上下游5 kb侧翼序列发现m Ping在日本晴和93-11中分别与23和3个已知基因相关联。本研究为阐明以m Ping的分布多样性为主要原因的粳稻和籼稻之间的遗传差异提供初步理论基础。     

Comparative Sequence Analysis of the <Emphasis Type="Italic">Phytochrome C</Emphasis> Gene and its Upstream Region in Allohexaploid Wheat Reveals New Data on the Evolution of its Three Constituent Genomes

Bread wheat is an allohexaploid with genome composition AABBDD. Phytochrome C is a gene involved in photomorphogenesis that has been used extensively for phylogenetic analyses. In wheat, the PhyC genes are single copy in each of the three homoeologous genomes and map to orthologous positions on the long arms of the group 5 chromosomes. Comparative sequence analysis of the three homoeologous copies of the wheat PhyC gene and of some 5 kb of upstream region has demonstrated a high level of conservation of PhyC, but frequent interruption of the upstream regions by the insertion of retroelements and other repeats. One of the repeats in the region under investigation appeared to have inserted before the divergence of the diploid wheat genomes, but was degraded to the extent that similarity between the A and D copies could only be observed at the amino acid level. Evidence was found for the differential presence of a foldback element and a miniature inverted-repeat transposable element (MITE) 5′ to PhyC in different wheat cultivars. The latter may represent the first example of an active MITE family in the wheat genome. Several conserved non-coding sequences were also identified that may represent functional regulatory elements. The level of sequence divergence (K_s) between the three wheat PhyC homoeologs suggests that the divergence of the diploid wheat ancestors occurred some 6.9 Mya, which is considerably earlier than the previously estimated 2.5–4.5 Mya. K_a/K_s ratios were <0.15 indicating that all three homoeologs are under purifying selection and presumably represent functional PhyC genes. RT-PCR confirmed expression of the A, B and D copies. The discrepancy in evolutionary age of the wheat genomes estimated using sequences from different parts of the genome may reflect a mosaic origin of some of the Triticeae genomes.     

A family of Tc1-like transposons from the genomes of fishes and frogs: evidence for horizontal transmission.

Tc1-like transposons are very widely distributed within the genomes of animal species. They consist of an inverted repeat sequence flanking a transposase gene with homology to the mobile DNA element, Tc1 of the nematode Caenorhabditis elegans. These elements seem particularly to infest the genomes of fish and amphibian species where they can account for 1% of the total genome. However, all vertebrate Tc1-like elements isolated so far are non-functional in that they contain multiple frameshifts within their transposase coding regions. Here I describe a Tc1-like transposon (PPTN) from the genome of a marine flatfish species (Pleuronectes platessa) which bears conserved inverted repeats flanking an apparently intact transposase gene. Closely related, although degenerate, Tc1-like transposons were also isolated from the genomes of Atlantic salmon (SSTN, Salmo salar) and frog (RTTN, Rana temporaria). Consensual nucleic acid sequences were derived by comparing several individual isolates from each species and conceptual amino acid sequences were thence derived for their transposases. Phylogenetic analysis of these sequences with previously isolated Tc1-like transposases shows that the elements from plaice, salmon and frog comprise a new subfamily of Tc1-like transposons. Each member is distinct in that it is not found in the genomes of the other species tested. Plaice genomes contain about 300 copies of PPTN, salmon 1200 copies of SSTN and frog genomes about 500 copies of RTTN. The presence of these closely related elements in the genomes of fish and frog species, representing evolutionary lines, which diverged more than 400 million years ago, is not consistent with a vertical transmission model for their distributions.     

Complete sequences of mitochondria genomes of Aedes aegypti and Culex quinquefasciatus and comparative analysis of mitochondrial DNA fragments inserted in the nuclear genomes

We present complete sequences of the mitochondrial genomes for two important mosquitoes, Aedes aegypti and Culex quinquefasciatus, that are major vectors of dengue virus and lymphatic filariasis, respectively. The A. aegypti mitochondrial genome is 16,655 bp in length and that of C. quinquefasciatus is 15,587 bp, yet both contain 13 protein coding genes, 22 transfer RNA (tRNA) genes, one 12S ribosomal RNA (rRNA) gene, one 16S rRNA gene and a control region (CR) in the same order. The difference in the genome size is due to the difference in the length of the control region. We also analyzed insertions of nuclear copies of mtDNA-like sequences (NUMTs) in a comparative manner between the two mosquitoes. The NUMT sequences occupy ~0.008% of the A. aegypti genome and ~0.001% of the C. quinquefasciatus genome. Several NUMTs were found localized in the introns of predicted protein coding genes in both genomes (32 genes in A. aegypti but only four in C. quinquefasciatus). None of these NUMT-containing genes had an ortholog between the two species or had paralogous copies within a genome that was also NUMT-containing. It was further observed that the NUMT-containing genes were relatively longer but had lower GC content compared to the NUMT-less paralogous copies. Moreover, stretches of homologies are present among the genic and non-genic NUMTs that may play important roles in genomic rearrangement of NUMTs in these genomes. Our study provides new insights on understanding the roles of nuclear mtDNA sequences in genome complexities of these mosquitoes.     

beta, a novel repetitive DNA element associated with tRNA genes in the pathogenic yeast Candida albicans

We have identified a novel 399 bp repetitive DNA element (which we designate beta  ) 9 bp upstream of a seryl-tRNA_CAG gene in the genome of Candida albicans . There are two copies of the seryl-tRNA_CAG gene, one on each homologue of chromosome VI, and the beta element is found upstream of one copy of the gene in C. albicans strain 2005E. The beta element is not present upstream of either copy of the seryl-tRNA_CAG gene in eight other laboratory strains of C. albicans tested, but was detected in this location in several fresh clinical isolates. Southern blot analysis indicated that there are approximately eight copies of the beta element per diploid C. albicans genome and that it is a mobile element, being present on at least two different chromosomes. Three unique genomic DNA clones containing the beta element were isolated from strain 2005E; in each case, a different tRNA gene was found immediately adjacent to the beta element. Three new tRNA genes from C. albicans have thus been identified: tRNA^Asp, tRNA^Ala and tRNA^Ile. The beta element shows no significant sequence homology to other known prokaryotic or eukaryotic repetitive elements, although an 8 bp repeat at the 3' end of the element is identical to that of the Ty3 retrotransposable element of Saccharomyces cerevisiae . We propose that the beta element is a solo long terminal repeat (LTR) sequence of a Ty3/gypsy-like transposable element in C. albicans that is closely associated with tRNA genes.