AFLP-Based Genetic Linkage Maps of the Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis> Thunberg

Amplified fragment length polymorphisms (AFLPs) were used for genome mapping in the Pacific oyster Crassostrea gigas Thunberg. Seventeen selected primer combinations produced 1106 peaks, of which 384 (34.7%) were polymorphic in a backcross family. Among the polymorphic markers, 349 were segregating through either the female or the male parent. Chi-square analysis indicated that 255 (73.1%) of the markers segregated in a Mendelian ratio, and 94 (26.9%) showed significant (P < 0.05) segregation distortion. Separate genetic linkage maps were constructed for the female and male parents. The female framework map consisted of 119 markers in 11 linkage groups, spanning 1030.7 cM, with an average interval of 9.5 cM per marker. The male map contained 96 markers in 10 linkage groups, covering 758.4 cM, with 8.8 cM per marker. The estimated genome length of the Pacific oyster was 1258 cM for the female and 933 cM for the male, and the observed coverage was 82.0% for the female map and 81.3% for the male map. Most distorted markers were deficient for homozygotes and closely linked to each other on the genetic map, suggesting the presence of major recessive deleterious genes in the Pacific oyster.     

Origin of new <Emphasis Type="Italic">Brassica</Emphasis> types from a single intergeneric hybrid between <Emphasis Type="Italic">B. rapa</Emphasis> and <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> by rapid chromosome evolution and introgression

Many novel lines were established from an intergeneric mixoploid between Brassica rapa (2n = 20) and Orychophragmus violaceus (2n = 24) through successive selections for fertility and viability. Pedigrees of individual F₂ plants were advanced to the 10th generation by selfing. Their breeding habit was self-compatible and different from the self-incompatibility of their female parent B. rapa, and these lines were reproductively isolated to different degrees from B. rapa and B. napus. The lines with high productivity showed not only a wide spectrum of phenotypes but also obvious variations in fatty acid profiles of seed oil and glucosinolate contents in seed meal. These lines had 2n = 36, 37, 38, 39 and 40, with 2n = 38 being most frequent (64.56%), and no intact O. violaceus chromosomes were detected by genomic in situ hybridization (GISH) analysis. Amplified fragment length polymorphism (AFLP) analyses revealed a high extent of variation in genomic compositions across all the lines. O. violaceus-specific bands, deleted bands in B. rapa and novel bands for two parents were detected in these lines, with novel bands being the most frequent. The morphological and genetic divergence of these novel types derived from a single hybrid is probably due to rapid chromosomal evolution and introgression, and provides new genetic resources for rapeseed breeding.     

Simple sequence repeat (SSR)-based gene diversity in <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis> and <Emphasis Type="Italic">Burkholderia mallei</Emphasis>

Pathogens Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) contain a large number (> 12,000) of Simple Sequence Repeats (SSRs). To study the extent to which these features have contributed to the diversification of genes, we have conducted comparative studies with nineteen genomes of these bacteria. We found 210 genes with characteristic types of SSR variations. SSRs with nonamer repeat units were the most abundant, followed by hexamers and trimers. Amino acids with smaller and nonpolar R-groups are preferred to be encoded by the variant SSRs, perhaps due to their minimal impacts to protein functionality. A majority of these genes appears to code for surface or secreted proteins that may directly interact with the host factors during pathogenesis or other environmental factors. There also are others that encode diverse functions in the cytoplasm, and this protein variability may reflect an extensive involvement of phase variation in survival and adaptation of these pathogens.     

Development and Characterization of EST-SSR Markers in the Eastern Oyster <Emphasis Type="Italic">Crassostrea virginica</Emphasis>

Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) in the eastern oyster (Crassostrea virginica). ESTs of the eastern oyster were downloaded from GenBank and screened for SSRs with at least eight units of dinucleotide or five units of tri-, tetra-, penta-, and hexa-nucleotide repeats. The screening of 9101 ESTs identified 127 (1.4%) SSR-containing sequences. Primers were designed for 88 SSR-containing ESTs with good and sufficient flanking sequences. Polymerase chain reaction (PCR) amplification was successful for 71 primer pairs, including 19 (27%) pairs that amplified fragments longer than expected sizes, probably due to introns. Sixty-six pairs that produced fragments shorter than 800 bp were screened for polymorphism in five oysters from three populations via polyacrylamide gels, and 53 of them (80%) were polymorphic. Fifty-three polymorphic SSRs were labeled and genotyped in 30 oysters from three populations via an automated sequencer. Five of the SSRs amplified more than two fragments per oyster, suggesting locus duplication. The remaining 48 SSRs had 2 alleles per individual, including 11 with null alleles. In the 30 oysters analyzed, the SSRs had an average of 9.3 alleles per locus, ranging from 2 to 24. Forty-three loci segregated in a family with 100 progeny, with nine showing significant deviation from Mendelian ratios (three after Bonferroni correction). Seventy percent of the loci were successfully amplified in C. rhizophorae and 34% in C. gigas. This study demonstrates that ESTs are valuable resources for the development of SSR markers in the eastern oyster, and EST-derived SSRs are more transferable across species than genomic SSRs.     

Evaluation of three molecular methods of repetitive element loci for differentiation of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> (MAP)

The aim of the present study is to evaluate the efficiency of three methods to determine the molecular diversity of 34 Mycobacterium avium subsp. paratuberculosis (MAP) strains isolated from 17 cattle herds. The applied methods included the analysis of sequence polymorphism of the mononucleotide (G1 and G2) and trinucleotide sequences (GGT) of the Short Sequence Repeats (SSR) and the determination of size polymorphism of 9 different Mycobacterial Interspersed Repetitive Units (MIRU) and 6 Variable Number Tandem Repeats (VNTR). Sequence analysis of SSR of 34 isolates showed 4, 6, and 2 alleles of G1, G2, and GGT repeats, respectively. The amplification of the investigated 9 MIRU units revealed only two discriminatory genotyping systems (MIRU2 and MIRU3). Out of 6 VNTR PCR differentiation methods, only one method could be recommended for genotyping purposes. The profile 7g-12g-4ggt-II-b-2 of the combination systems G1-G2-GGT-MIRU2-MIRU3-VNTR1658 dominates among the examined isolates and was detected in 14.7% of the isolates. The use of certain repetitive loci of SSR, MIRU, and VNTR techniques in this study showed greater potential than others for the characterization of MAP isolates. The recommended loci can be used for the epidemiological tracing of MAP field strains and to determine the relationships between isolates in different herds.     

Cloning and characterization of a bile salt hydrolase (<Emphasis Type="Italic">bsh</Emphasis>) from <Emphasis Type="Italic">Bifidobacterium adolescentis</Emphasis>

A gene coding for bile salt hydrolase (BSH) from Bifidobacterium adolescentis was cloned and expressed in Escherichia coli, and the nucleotide sequence was determined. The BSH of E. coli transformants was produced intracellularly in the absence of bile salts. A unique bsh promoter (P_bsh) sequence was identified by using a Neural Network Promoter Prediction (NNPP, version 2.2). In spite of their high-level sequence homology with other bsh genes in the Bifidobacterium species, their genetic organization surrounding the bsh gene and their promoter sequences are different depending on the species.     

Quantification of<Emphasis Type="Italic">Bacillus</Emphasis> species in a wastewater treatment system by the molecular analyses

Bacillus species were observed and quantified by molecular approaches, using the 165 rDNA primers/probes, in a wastewater treatment plant designed for the purpose of stimulating the growth ofBacillus species. The plant has been operating as a test plant since 1997 in the city of Ina, Japan, with excellent treatment performance. Observations byin situ hybridization, usingBacillus-specific probes, indicated thatBacillus strains were inhabited in the plant and their numbers decreased during the treatment process. Similar results were obtained from a quantitative PCR analysis using aBacillus-specific primer set, and the amount of DNA originating from variousBacillus species was maximally 1.91% of the total DNA in the wastewater treatment tank. Clone library analysis using theBacillus-specific primers suggested that, while the population was noticeably increased, the phylogenetic diversity of the increasingBacillus species was very low.     

Intramolecular G-quadruplexes formed by d(GT)<Subscript>12</Subscript> microsatellite sequence in the presence of K<Superscript>+</Superscript>

Polymorphic d(GT) _n microsatellite sequences are known to dramatically affect the regulation of gene expression. CD spectroscopy, UV melting, fluorescence polarization of ethidium bromide (EtBr), and FRET allowed the detection of a new G-quartet fold formed by the d(GT)₁₂ oligonucleotide in 0.01 M Na-phosphate (pH 8.0) in the presence of 0.1 M KCl. The monomolecular type of the structure was verified by measuring the rotational relaxation time (ρ = 28 ± 0.5 ns) of the EtBr: d(GT)₁₂ complex. CD spectra supported the G-quartet formation. FRET was used to estimate the distance between intercalated EtBr and FITC covalently attached to the 5′ end of d(GT)₁₂ (R ≤ 17 Å). The experimental data agree with the self-folding of d(GT)₁₂ in a G-quartet structure.     

Conserved 5S and variable 45S rDNA chromosomal localisation revealed by FISH in <Emphasis Type="Italic">Astyanax scabripinnis</Emphasis> (Pisces,Characidae)

Fluorescence in situhybridisation (FISH) and double FISH experiments were carried out to ascertain the chromosomal distribution pattern of the 45S and 5S ribosomal (r) DNAs in four populations of the characid fish Astyanax scabripinnis – a group considered to be a species complex for its wide karyotypical and morphological diversity. The results regarding the 45S rDNA agreed with this hypothesis, since these sites showed intra- and inter-populational, numerical and positional variations. However, the data obtained with the 5S rDNA probe revealed a highly conserved chromosomal distribution pattern of these sequences among individuals of each population, as well as among the populations analysed. We consider this contrasting situation as a functional divergence between 45S and 5S ribosomal DNAs, which may reflect the localisation of these sequences in distinct nuclear compartments, leading them to undergo differentiated evolutionary processes.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Monitoring of algicidal bacterium, <Emphasis Type="Italic">Alteromonas</Emphasis> sp. Strain A14 in its application to natural <Emphasis Type="Italic">Cochlodinium polykrikoides</Emphasis> blooming seawater using fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization

The red tide of dinoflagellate, Cochlodinium polykrikoides has frequently occurred in coastal waters, causing severe damage to fisheries. In the present study, the algicidal bacterium Alteromonas sp. A14 isolated from the southern coast of Korea was applied to a red tide of C. polykrikoides in a laboratory experiment. In the experiment, the abundance of the strain A14 was monitored using fluorescence in situ hybridization. Inoculation of the A14 at a final cell density of 9.0 x 10(5) cells/ml caused a significant decrease in C. polykrikoides abundance from 1,830 to 700 cells/ml during 2 days, while abundances of harmless diatoms rapidly increased from 3 days. Abundances of both A14 and other bacteria increased to 1 day. After 1 day, with flagellate abundance increased, bacterial abundance decreased. Finally, algicidal bacterial abundance decreased to 3.5 x 10(4) cells/ml. In the biological control of harmful algal blooms, in addition to decrease in target algal abundance and not occurrence of other harmful blooms, decrease in abundance of utilized organism is also important. This study emphasizes the importance of monitoring the inoculated bacterium when applying bacterium to natural seawater.     

Karyotypic characterization and genomic organization of the 5S rDNA in <Emphasis Type="Italic">Polypterus senegalus</Emphasis> (Osteichthyes,Polypteridae)

Polypteridae (Cladistia) is a family of archaic fishes, confined to African freshwaters. On account of their primitiveness in anatomical and morphological characters and mosaic relationships among lower Osteichthyans fishes, they constitute an important subject for the study of evolution in vertebrates. Very little is known about the karyological structure of these species. In this article, a cytogenetic analysis on twenty specimens of Polypterus senegalus (Cuvier, 1829) was performed using both classical and molecular techniques. Karyotype (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG) _n , (GATA)₇ repeats and ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA₃ staining and FISH. Staining with Ag-NOR showed the presence of two GC rich NORs on the p arm of the chromosome pair no. 1. CMA₃ marked all centromerical and some (no. 1 and no. 14) telomeric regions. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair no. 14. FISH with 18S rDNA marked the telomeric region of the p arm of the chromosome pair no. 1, previously marked by Ag-NOR. (GATA)₇ repeats marked the subtelomeric regions of all chromosome pairs, with the exclusion of the no. 1, 3 and 14. Hybridization with telomeric probes (TTAGGG) _n showed bright signals at the end of all chromosomes. After cloning, the 5SrDNA alignment revealed an organization of sequences made up of two different classes of tandem arrays (5S type I and 5S type II) of different lengths.     

IS<Emphasis Type="Italic">Rm31</Emphasis>, a new insertion sequence of the IS<Emphasis Type="Italic">66</Emphasis> family in<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti pSymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, ISRm31. ISRm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that ISRm31 is a member of the IS66 family. ISRm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying ISRm31 suggested that ISRm31 inserts randomly into S. meliloti genomes. Moreover, analysis of ISRm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, ISRm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.     

New PCR based markers allowed to identify <Emphasis Type="Italic">Secale</Emphasis> chromatin in wheat-<Emphasis Type="Italic">Secale africanum</Emphasis> introgression lines

The genus of Secale has many agronomically important characters. In order to use the best of this species, markers tracking the rye chromatin incorporated into wheat must be developed. In this study, one rye genome-specific random amplified polymorphic DNA (RAPD) marker was isolated from Secale africanum (R^a genome). Two cloned markers, named OPP13₁₁₆₅ and OPP13₆₆₂, were 1165 bp and 662 bp, respectively. Sequence analysis revealed that OPP13₁₁₆₅ was highly homologous to a part of a new class of transposon-like gene called the Revolver family, and OPP13₆₆₂ was partially similar to LTR gypsy-like retrotransposon. Fluorescence in situ hybridization (FISH) showed only OPP13₁₁₆₅ localized within the whole arms of rye except their terminal regions and no signal was detected on wheat chromosomes, while OPP13₆₆₂ had no hybridization signal detected on wheat and rye genomes. Based on these sequences, two pairs of sequence-characterized amplified region (SCAR) primers were designed, and the resulted SCAR markers were able to target both cultivated and wild Secale species. The FISH patterns and the two SCAR markers should be able to identify and track all wheat-rye translocation lines, especially the S. africanum chromatin.     

Repeated losses of TTAGG telomere repeats in evolution of beetles (Coleoptera)

We studied the occurrence of (TTAGG) _n telomere repeats in 12 species of beetles, representing main lineages of the Coleoptera phylogenetic tree, by Southern hybridization and fluorescence in situ hybridization (FISH). In contrast to other insect orders, beetles were heterogeneous with respect to the occurrence of TTAGG repeats. In addition, the presence or absence of (TTAGG) _n motif was irrespective of phylogenetic relationships. In the suborder Polyphaga, six species displayed positive hybridization signals. These were Silpha obscura, Agrilus viridis, Ampedus sanguineus, Stegobium paniceum, Oryzaephilus surinamensis, and Leptinotarsa decemlineata. Whereas negative signals were obtained in three polyphagan species, Geotrupes stercorarius, Thanasimus formicarius, and Sitophilus granarius. In the suborder Adephaga, the TTAGG sequence was present in one species, Graphoderus cinereus, and absent in two species, Orectochilus villosus and Pterostichus oblongopunctatus. We concluded that the telomerase-dependent (TTAGG) _n motif had been repeatedly lost in different phylogenetic branches of Coleoptera and probably replaced with another mechanism of telomere elongation. This had to happen at least 5–6 times. The results suggest a predisposition or a backup mechanism of telomere maintenance in the genome of beetles that enabled them to make frequent evolutionary changes in the telomere composition.     

A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.