bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative evaluation of within-cultivar variation of rice (Oryza sativaL.) using microsatellite and RFLP markers
Authors: Olufowote Johnson O. , Xu Yunbi , Chen Xiuli , Goto Mak , McCouch Susan R. , Park William D. , Beachell Henry M. , Dilday Robert H.
Published: May 2008

The objective of this study was to determine an efficient way of detecting within-cultivar variation in rice varieties obtained from national and international germplasm collections. Seventy-one rice cultivars were evaluated for within-cultivar variation using a combination of phenotypic, RFLP, and microsatellite or simple sequence length polymorphism (SSLP). Variation between individuals within an accession and between duplicate accessions within a cultivar was detected even in cultivars that had been purified by phenotypic evaluation. Landrace cultivars were more heterogeneous and displayed a larger number of both RFLP and SSLP alleles than did modern cultivars. Microsatellite markers detected a greater number of alleles and were able to discriminate between even closely related individuals more efficiently than RFLPs. Some microsatellite markers were more informative than others for assessing genetic diversity. Single markers revealed 5.6–61.1% of the total variation detected by the 10 SSLP markers. Some marker combinations were complementary, providing more information than others. Several combinations of 4 SSLP markers detected as much as 94% of the total within-cultivar variation detected by the 10 SSLP markers. These results suggest that the use of four well-chosen microsatellites would be an efficient method for evaluating the heterogeneity of rice accessions.Key words: genetic variation, RFLP, microsatellite markers, simple sequence length polymorphism, SSLP, rice.
Physical mapping of ribosomal DNA sites in Festuca arundinacea and related species by in situ hybridization
Authors: Thomas H. M. , Harper J. A. , Meredith M. R. , Morgan W. G. , King I. P.
Published: May 2008

The positions of the 18S–5.8S–26S and 5S rRNA genes have been physically mapped on the chromosomes of diploid, tetraploid, and hexaploid Festuca species by in situ hybridization. The number and position of the rDNA sites in the species were compared. The results confirm some of the earlier phylogenetic studies of these species but suggest that some structural rearrangements have occurred and that sites have been lost during polyploidization. Keywords: Festuca, in situ hybridization, phylogeny, physical mapping, rDNA.
Identification and characterization of microsatellites in Norway spruce (Picea abiesK.)
Authors: Pfeiffer Antonella , Olivieri Angelo M. , Morgante Michele
Published: May 2008

Norway spruce (Picea abies) genomic libraries were screened for presence of dinucleotide AC/GT and AG/CT microsatellites (or simple sequence repeats). On average, one (AG)nmicrosatellite every 194 kb and one (AC)nmicrosatellite every 406 kb were found. Forty-six positive clones were sequenced and primers flanking 24 AG microsatellites and 12 AC microsatellites designed. Only seven (20%) of them produced the expected single-locus polymorphic pattern when used to amplify Norway spruce DNAs. The other primer pairs gave either multiple bands or bad amplification, or a single monomorphic fragment. Such a small proportion of successful primer pairs was attributed to the high level of complexity of the Norway spruce genome. Dot blot analysis of the clones showed that many of them contained repetitive DNA and that those giving the single-locus polymorphic patterns usually corresponded to single-copy sequences. A family of repetitive DNA that contained AG repeats was identified and was present in about 40 000 copies per haploid genome. Simple Mendelian inheritance was observed for all the polymorphisms tested. The average number of alleles was 13, ranging from 6 to 22, and the expected heterozygosity was 0.79 when seven microsatellites were used to genotype a panel of 18 trees representing different populations. Compared with isozymes, microsatellites are about five times more informative and could provide an extremely valuable source of markers for genome mapping and genetic diversity studies.Key words: microsatellite, repetitive DNA, hypervariability, Picea abies, genome complexity.
BAC contig development by fingerprint analysis in soybean
Authors: Marek L. Fredrick , Shoemaker R. C.
Published: May 2008

We constructed a soybean bacterial artificial chromosome (BAC) library suitable for map-based cloning and physical mapping in soybean. This library consists of approximately 40 000 clones (4–5 genome equivalents) stored individually in 384-well microtiter dishes. A random sampling of 224 clones yielded an average insert size of 150 kb, giving a 98% probability of recovering any specific sequence. We screened the library for seven single or very low copy genie or genomic sequences using the polymerase chain reaction (PCR) and found between one and seven BACs for each of the seven sequences. When testing the library with a portion of the soybean psbA chloroplast gene, we found less than 1% chloroplast DNA representation. We also screened the library for eight different classes of disease resistance gene analogs (RGAs) and identified BACs containing all RGAs except class 8. We arranged nine of the class 1 RGA BACs and six of the class 3 RGA BACs into individual contigs based on fingerprint patterns observed after Southern probing of restriction digests of the member BACs with a class-specific sequence. This resulted in the partial localization of the different multigene family sequences without precise definition of their exact positions. Using PCR-based end rescue techniques and RFLP mapping of BAC ends, we mapped individual BACs of each contig onto linkage group J of the soybean public map. The class 1 contig mapped to the region on linkage group J that contains several disease resistance genes. The class 1 contig extended approximately 400 kb. The arrangement of the BACs within this contig has been confirmed using PCR. One end of the class 1 contig core BAC mapped to two positions on linkage group J and cosegregated with two class 1 RGA loci, suggesting that this segment is within an area of regional duplication.Key words: bacterial artificial chromosome, BAC library, soybean, contig, resistance gene analog.
Simple sequence repeats in watermelon (Citrullus lanatus(Thunb.) Matsum. &Nakai)
Authors: Jarret R. L. , Merrick L. C. , Holms T. , Evans J. , Aradhya M. K.
Published: May 2008

Simple sequence repeat length polymorphisms were utilized to examine genetic relatedness among accessions of watermelon (Citrullus lanatus (Thunb.) Matsum. &Nakai). A size-fractionated TaqI genomic library was screened for the occurrence of dimer and trimer simple sequence repeats (SSRs). A total of 96 (0.53%) SSR-bearing clones were identified and the inserts from 50 of these were sequenced. The dinucleotide repeats (CT)nand (GA)naccounted for 82% of the SSRs sequenced. PCR primer pairs flanking seven SSR loci were used to amplify SSRs from 32 morphologically variable watermelon genotypes from Africa, Europe, Asia, and Mexico and a single accession of Citrullus colocynthis from Chad. Cluster analysis of SSR length polymorphisms delineated 4 groups at the 25% level of genetic similarity. The largest group contained C. lanatus var. lanatus accessions. The second largest group contained only wild and cultivated "citron"-type or C. lanatus var. citroides accessions. The third group contained an accession tentatively identified as C. lanatus var. lanatus but which perhaps is a hybrid between C. lanatus var. lanatus and C. lanatus var. citroides. The fourth group consisted of a single accession identified as C. colocynthis. "Egusi"-type watermelons from Nigeria grouped with C. lanatus var. lanatus. The use of SSRs for watermelon germplasm characterization and genetic diversity studies is discussed.Key words: Citrullus, watermelon, simple sequence repeats, genetic diversity.
A strategy for detecting chromosome-specific rearrangements in rye
Authors: Alvarez E. , Alonso-Blanco C. , Suárez R. García , Ferreira J. J. , Roca A. , Giraldez R.
Published: May 2008

To obtain translocations involving specific chromosomes in rye, a line in which chromosome 1R has large C-bands on its two telomeres but which lacks C-bands (or has very small ones) on the telomeres of the remaining chromosomes was used. About 6% of the plants produced using pollen from irradiated (1.2 krad (1 rad = 10 mGy)) spikes of this line possessed structural changes involving the labeled chromosome. These aberrations included translocations, ring chromosomes, isochromosomes, and telocentrics. It is concluded (i) that all nonlabeled chromosomes have the same probability of participating in reciprocal translocations with the labeled chromosome, 1R, and (ii) that most induced reciprocal translocations involved exchanges of chromosome segments of approximately equal length. The use of lines having the appropriate combination of telomeric C-bands improves the efficiency of obtaining reciprocal translocations involving specific chromosomes that could be used in the construction of detailed physical maps.Key words: Secale, translocations, C-banding, x-ray irradiation.
Genetic diversity among a complex of cereal cyst nematodes inferred from RFLP analysis of the ribosomal internal transcribed spacer region
Authors: Bekal S. , Gauthier J. P. , Rivoal R.
Published: May 2008

This study examined the restriction polymorphism (RFLP) of the nuclear ribosomal DNA in Heterodera avenae, H. filipjevi, H. mani, H. latipons, and the taxonomically unclear Gotland strain in order to establish a molecular characterization and phylogenetic relationships in the complex of cereal cyst nematodes (CCN). The internal transcribed spacer (ITS) and 5.8S rDNA were amplified by PCR from a single female or a cyst of 27 different geographic isolates of the CCN complex and one population of H. schachtii, used as outgroup. The amplified product was 1.2 kb long and 14 of 15 enzymes produced restriction fragments for each isolate. Relationships between populations were determined from UPGMA analysis based on distance values calculated from RFLP data. Digestions with TaqI clearly differentiated H. avenae, H. latipons, and a group composed of H. filipjevi and the Gotland strain. Six endonucleases (HaeIII, HinfI, ItaI, PstI, TaqI, and Tru9I) produced the same restriction pattern with H. filipjevi and the Gotland strain, and both were clearly separated from H. avenae with PstI. Restriction sites have revealed a mixture of the species H. latipons and H. avenae, and possible infraspecific variation in H. avenae. The inferred phylogenetic relationships of species in the CCN complex are in agreement with their morphological characterization.Key words: cereal cyst nematodes, Heterodera avenae, PCR, RFLP, ribosomal diversity.
Molecular phylogeny of mangroves II. Intra -and inter-specific variation in Avicennia revealed by RAPD and RFLP markers
Authors: Parani M. , Lakshmi M. , Elango S. , Ram Nivedita , Anuratha C. S. , Parida Ajay
Published: May 2008

Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were used to estimate intra- and inter-specific variation in three species of an exclusive mangrove genus, Avicennia. Intrapopulation polymorphism among the 10 populations of Avicennia marina, as measured by percentage of polymorphic RAPDs, varied between 17.8 and 38.9%, with a standard deviation of 7.28, and the coefficient of variation was 26.5%. Polymorphism in Avicennia officinalis (Pichavaram population, 32.3%) and Avicennia alba (Coringa population, 37.8%) was greater than the intrapopulation variation observed in the populations of A. marina from each of the respective locations. It was greater than the average percentage of polymorphism at the intrapopulation level (27.47%) but far less than the variation measured at the interpopulation level in A. marina. Interpopulation variation in A. marina (76.7% for RAPDs and 66% for RFLPs) was greater than the variation in any individual population of this species, indicating a high degree of divergence between the populations. Interpopulation variation as revealed by RAPD and RFLP markers did not indicate the existence of more than one distinct entity in this species in India. The implications of these observations in genetic sampling and conservation are discussed. Statistical analysis of 109 RAPDs and 84 RFLPs observed in one representative genotype from each species showed that the widely distributed A. marina was more closely related to A. alba (genetic distance (1 − F) = 0.22) than to A. officinalis (genetic distance (1 − F) = 0.37). RAPD analysis of six randomly selected genotypes in each species and principal component analysis of the data also favoured this observation.Key words: mangrove, Avicennia, molecular markers, genetic diversity, conservation.
Estimating genetic variation in sugar beets and wild beets using pools of individuals
Authors: Kraft Thomas , Säll Torbjörn , Fridlund Birgitta , Hjerdin Annika , Tuvesson Stig , Halldén Christer
Published: May 2008

The study describes the genetic structure in sugar beets and in wild beets (Beta vulgaris) using 30 RFLP markers. Samples consisting of pooled plant material of 100 individuals from each line and population were used to analyse 120 sugar beet breeding lines and 91 wild beet populations. Greater variation was found among the wild populations than among the breeding lines. Although the two major groups of breeding lines, monogerm and multigerm, had approximately equal amounts of genetic variation, in the monogerm group more of this variation was partitioned among the lines than within the lines. Furthermore, despite most of the variation being shared by the two groups, the two groups were found to be separated along the first two components in a principal component analysis. Computer simulations were carried out to evaluate the usefulness of the pooled-sample strategy employed in the investigation. These simulations showed the use of pooled samples to be a better alternative than that of analysing a few plants individually.Key words: Beta vulgaris, genetic variation, RFLP, pooled samples.
Biolistic transformation of haploid isolated microspores of barley (Hordeum vulgare L.)
Authors: Yao Qing A. , Simion Ecaterina , William Manilal , Krochko Joan , Kasha Ken J.
Published: May 2008

Transgenic barley plants were produced by the direct delivery of plasmid DNA into isolated microspores of barley cv. Igri using high velocity microprojectiles. The plasmid pAHC25 contained the uidA and bar genes, each under the control of a maize Ubi1 promoter. Bombarded microspores were cultured and selected on solid medium containing varying concentrations (2–5 mg/L) of the Basta herbicide active agent bialaphos. The effectiveness of selection with bialaphos depended on its interaction with the medium component glutamine. Six transgenic plants (R0) were obtained, and the presence of the uidA and bar genes and their integration into nuclear DNA in transformed R0 plants were confirmed by PCR and Southern blot analysis. Phosphinothricin acetyltransferase activity was observed in all six R0 transgenic plants, whereas none showed β-glucuronidase (GUS) activity in histochemical GUS assays. Two of the six R0 plants were haploid and sterile; one of them was trisomic and partially sterile; the remainder were diploid, but one of them was also sterile. Inheritance of the transgenes in progeny of three seed-producing transgenic plants was investigated. Southern blot analysis of genomic DNA from R1 plants showed that the introduced bar and uidA genes were hemizygous and stably cotransmitted to the R1 progeny derived from self-pollination. Analysis of Basta resistance and the integration of the bar gene by PCR analysis in R1 plants indicated that the bar gene was being inherited and expressed as a single dominant trait. Fluorescent in situ hybridization was performed on chromosomes of the trisomic plant to confirm the presence of transgenes in the genome.Key words: barley, microspore, biolistic transformation, bialaphos, haploid, FISH.
Comparative analysis of genetic relationships in barley based on RFLP and RAPD markers
Authors: Noli Enrico , Salvi Silvio , Tuberosa Roberto
Published: May 2008

Genetic relationships have seldom been analyzed with different types of molecular markers in order to compare the information provided by each marker class. We investigated genetic relationships among nine barley cultivars using separate cluster analyses based on restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs). Genomic DNA restricted with three enzymes and hybridized with 68 probes revealed 415 RFLPs (74.2% of all bands). Among the 128 primers used for RAPD analysis, 100 provided a reproducible profile, 89 of which revealed 202 polymorphic and 561 monomorphic bands (26.5 and 73.5%, respectively). A nonrandom distribution of 62 RAPDs with a tendency to cluster near centromeric regions was produced when these RAPDs were mapped using 76 doubled-haploid lines derived from a cross between two of the nine cultivars. The correlation between the RFLP and RAPD similarity matrices computed for the 36 pairwise comparisons among the nine cultivars was equal to 0.83. The dendrograms obtained by cluster analyses of the RFLP and RAPD data differed. These results indicate that in barley the information provided by RFLPs and RAPDs is not equivalent, most likely as a consequence of the fact that the two marker classes explore, at least in part, different portions of the genome.Key words: Hordeum vulgare L., genetic distance, molecular markers, cluster analysis.
Characterization and genomic organization of Ty1-copiagroup retrotransposons in rye (Secale cereale)
Authors: Pearce Stephen R. , Harrison Gill , Heslop-Harrison Pat (J. S.) , Flavell Andrew J. , Kumar Amar
Published: May 2008

The genomic organisation of the Ty1-copia retrotransposons in rye (Secale cereale) has been studied. We have used the polymerase chain reaction (PCR) to amplify sequences from a conserved domain of the reverse transcriptase gene of the Ty1-copia retrotransposons in this species. Sequence analysis of 26 of these PCR products shows them to be a highly heterogeneous population, a feature that is common in plants. Slot blot analysis shows that there are about 100 000 individual Ty1-copia retrotransposons in rye. In situ hybridization of a heterogeneous probe, representing the whole population of rye Ty1-copia retrotransposon sequences, to chromosome spreads of triticale (×Triticosecale), a rye–wheat hybrid, shows that these sequences are present throughout all the rye chromosomes but absent from the centromeric regions and, in particular, from the terminal heterochromatin. Southern analysis of oat, barley, wheat, and rye, using as a probe R9, one of the rye sequences that is closely similar to the BARE-1 element of barley, shows that close relatives of this retrotransposon subgroup are present in all these species in high copy number. Northern analysis on RNAs from seedlings shows that the BARE-1 subgroup is transcribed in all these cereal plants but in variable amounts: high in barley, moderate in wheat and rye, and extremely low in oat.Key words: retrotransposons, Secale cereale, plant genome, Ty1-copia, in situ hybridization.
Use of a subset of doubled-haploid lines for RAPD interval mapping in barley
Authors: Dahleen L. S. , Hoffman D. L. , Dohrmann J. , Gruber R. , Franckowiak J.
Published: May 2008

Molecular markers have been used in barley to locate genes and quantitative trait loci. Only a few RAPD markers have been located on barley marker maps. The objectives of this study were (i) to place RAPD markers in specific intervals on the barley linkage map developed from the cross Steptoe (S) × Morex (M), (ii) to examine the distribution of RAPD markers, and (iii) to compare markers amplified by Taq DNA polymerase with those amplified by the Stoffel fragment of Taq DNA polymerase. Screening of DNA from S and M with 362 decamer primers identified 85 that amplified 127 reliable RAPDs. A subset of 15 doubled-haploid (DH) lines from the 150 DH line mapping population was used to place these RAPD markers in intervals on the SM map. This subset can be used for rapid placement of any new markers on the SM linkage map. Most of the RAPD markers were dominant but four codominant RAPDs were identified. The RAPDs were not evenly distributed, with many clustered around the centromeric region of each chromosome. Two of these clusters were located in intervals larger than 15 cM. Testing of 38 to 42 additional DH lines provided more precise placement of eight of the markers in these clusters. Reliable RAPDs were detected with 44% of the primers tested with the Stoffel fragment, but with only 17% of the primers tested with Taq DNA polymerase. These RAPDs provide additional markers for use in barley improvement.Key words: Hordeum vulgare, interval mapping, molecular markers.
Root tip cell cycle synchronization and metaphase-chromosome isolation suitable for flow sorting in common wheat (Triticum aestivum L.)
Authors: Lee Jai-Heon , Arumuganathan K. , Yen Y. , Kaeppler S. , Kaeppler H. , Baenziger P. S.
Published: May 2008

An efficient procedure for cell-cycle synchronization in meristematic root tips was achieved in common wheat. Treatment parameters for synchronizing the cell cycle of root tip meristem cells, such as time-course and applied concentrations of various chemicals, were systematically tested and optimized by flow cytometric analysis of isolated nuclei. High mitotic indices (69.5% in the root tip meristematic area) were routinely obtained by treating germinating seeds with 1.25 mM hydroxyurea for 16 h, followed by incubation in a hydroxyurea-free solution for 2 h, and treatment with 1 μM trifluralin for 4 h. Uniform seed germination prior to treatment is very important for achieving consistently high metaphase indices in the root tips. Large numbers of metaphase chromosomes, suitable for flow cytometric analysis and sorting, were isolated from synchronized root tip cells. Flow sorted wheat chromosomes, via univariate and bivariate analysis, showed four major chromosome peaks. Each discrete peak may represent wheat chromosome types with similar DNA content. Bivariate flow karyotyping based on AT and GC content did not improve the separation of wheat chromosomes.Key words: flow cytometry, trifluralin, hydroxyurea, univariate analysis, bivariate analysis.
Physical localisation of repetitive DNA sequences in Alstroemeria: karyotyping of two species with species-specific and ribosomal DNA
Authors: Kamstra Silvan A. , Kuipers Anja G. J. , Jeu Marjo J. De , Ramanna M. S. , Jacobsen Evert
Published: May 2008

Fluorescence in situ hybridization (FISH) was used to localise two species-specific repetitive DNA sequences, A001-I and D32-13, and two highly conserved 25S and 5S rDNA sequences on the metaphase chromosomes of two species of Alstroemeria. The Chilean species, Alstroemeria aurea (2n = 16), has abundant constitutive heterochromatin, whereas the Brazilian species, Alstroemeria inodora, has hardly any heterochromatin. The A. aurea specific A001-I probe hybridized specifically to the C-band regions on all chromosomes. The FISH patterns on A. inodora chromosomes using species-specific probe D32–13 resembled the C-banding pattern and the A001-I pattern on A. aurea chromosomes. There were notable differences in number and distribution of rDNA sites between the two species. The 25S rDNA probe revealed 16 sites in A. aurea that closely colocalised with A001-I sites and 12 in A. inodora that were predominantly detected in the centromeric regions. FISH karyotypes of the two Alstroemeria species were constructed accordingly, enabling full identification of all individual chromosomes. These FISH karyotypes will be useful for monitoring the chromosomes of both Alstroemeria species in hybrids and backcross derivatives.Key words: Alstroemeria, fluorescence in situ hybridization, FISH, repetitive DNA, ribosomal DNA, karyotype.
Map-based cloning of a gene sequence encoding a nucleotide-binding domain and a leucine-rich region at the Cre3 nematode resistance locus of wheat
Authors: Lagudah Evans S. , Moullet Odile , Appels Rudi
Published: May 2008

The Cre3 gene confers a high level of resistance to the root endoparasitic nematode Heterodera avenae in wheat. A DNA marker cosegregating with H. avenae resistance was used as an entry point for map-based cloning of a disease resistance gene family at the Cre3 locus. Two related gene sequences have been analysed at the Cre3 locus. One, identified as a cDNA clone, encodes a polypeptide with a nucleotide binding site (NBS) and a leucine-rich region; this member of the disease resistance gene family is expressed in roots. A second Cre3 gene sequence, cloned as genomic DNA, appears to be a pseudogene, with a frame shift caused by a deletion event. These two genes, related to members of the cytoplasmic NBS – leucine rich repeat class of plant disease resistance genes were physically mapped to the distal 0.06 fragment of the long arm of wheat chromosome 2D and cosegregated with nematode resistance.Key words: cereal cyst nematode, disease resistance genes, nucleotide-binding site, leucine-rich repeat.
Chiasma redistribution in the presence of different sized supernumerary segments in a grasshopper: dependence on nonhomologous synapsis
Authors: Cerro A. L. del , Santos J. L.
Published: May 2008

Eight different sized supernumerary segments located at distal ends of the long arms of chromosomes M4, M5, M6, and S8 of the grasshopper Stenobothrus festivus were studied in males with regard to the synaptic process and chiasma distribution in the bivalents that carry them. The M4, M5, and M6 bivalents heterozygous for extra segments were always monochiasmate, in contrast to their bichiasmate condition observed in basic homozygotes. Furthermore, the presence of any of these extra segments led to chiasma redistribution in the carrier bivalents, so that such chiasmata were formed preferentially further away from the extra segment. The intensity of this effect is dependent on the size of the segment. Not all heteromorphic bivalents exhibited synaptonemal complexes with equalized axes at pachytene, but there was always a variable proportion of heterosynapsis around the distal ends of the long arms that was dependent on both the size of the segment and the size of the carrier chromosome. It is proposed that the absence of chiasmata in nonhomologous synapsed regions is responsible for the results obtained. Length measurements of the different extra segments and their carrier chromosomes between pachytene and diplotene indicated that synaptonemal complex is underrepresented in supernumerary heterochromatin.Key words: chiasma distribution, grasshopper, heterosynapsis, supernumerary segment, synaptonemal complex.
Molecular marker based taxonomy and phylogeny of Guinea yam (Dioscorea rotundata–D.cayenensis)
Authors: Ramser Juliane , Weising Kurt , Terauchi Ryohei , Kahl Günter , Lopez-Peralta Cristina , Terhalle Werner
Published: May 2008

Four different molecular techniques were used to assess relationships among 21 accessions of Guinea yam (Dioscorea rotundata and Dioscorea cayenensis) and 21 accessions belonging to seven putative progenitor species. Random amplified polymorphic DNA (RAPD) and microsatellite-primed PCR (MP-PCR) analysis yielded 246 informative characters that were transformed into a matrix of pairwise distances and analyzed by neighbor joining or split decomposition. Both methods gave congruent results. Well-separated groups were formed that corresponded to their species designation. Dioscorea rotundata and D. cayenensis accessions were clearly separated from each other, supporting the concept that both are distinct species. Two morphological intermediates grouped together with D. rotundata. All investigated species fell into two main clusters, one comprising D. rotundata, D. cayenensis, Dioscorea abyssinica, Dioscorea liebrechtsiana, and Dioscorea praehensilis, the other comprising Dioscorea smilacifolia, Dioscorea minutiflora, Dioscorea burkilliana, and Dioscorea togoensis. The same grouping was also obtained by comparative sequence analysis of chloroplast DNA, which supports earlier studies of nuclear rDNA variation and chloroplast restriction fragment length polymorphisms. We also analyzed the same set of Dioscorea samples with the recently developed random amplified microsatellite polymorphism (RAMPO) technique. A series of diagnostic RAMPO bands was identified that clearly distinguished between D. rotundata and D. cayenensis. Some of these bands could also be traced back to the putative progenitors of both species. The evolutionary origin of Guinea yam is discussed in light of the present results.Key words: random amplified polymorphic DNA, RAPD, random amplified microsatellite polymorphisms, RAMPO, chloroplast DNA, genetic relatedness.
Molecular analysis of a NOR site polymorphism in brown trout (Salmo trutta): organization of rDNA intergenic spacers
Authors: Castro Jaime , Sánchez Laura , Martínez Paulino , Lucchini Stefania De , Nardi Irma
Published: May 2008

Using restriction endonuclease mapping, we have analyzed the organization of rDNA (DNA coding for ribosomal RNA (rRNA)) units in the salmonid fish Salmo trutta, as an initial step toward understand the molecular basis of a nucleolar organizer region (NOR) site polymorphism detected in this species. The size of the rDNA units ranged between 15 and 23 kb, with remarkable variation both within individuals and between populations. Three regions of internal tandem repetitiveness responsible for this length polymorphism were located to the intergenic spacers. NOR site polymorphic individuals showed a higher number of length classes, in some cases forming a complete 1 kb fragment ladder. The amount of rRNA genes was as much as 8-fold higher in polymorphic individuals compared with standard individuals. All individuals from the most polymorphic population showed a 14-kb insertion of unknown nature in a small proportion (below 25%) of the 28S rRNA genes.Key words: rRNA genes, repetitive elements, molecular organization, rDNA amount.
Identification, genetic localization, and allelic diversity of selectively amplified microsatellite polymorphic loci in lettuce and wild relatives (Lactucaspp.)
Authors: Witsenboer H. , Michelmore R. W. , Vogel J.
Published: May 2008

Selectively amplified microsatellite polymorphic locus (SAMPL) analysis is a method of amplifying microsatellite loci using generic PCR primers. SAMPL analysis uses one AFLP primer in combination with a primer complementary to microsatellite sequences. SAMPL primers based on compound microsatellite sequences provided the clearest amplification patterns. We explored the potential of SAMPL analysis in lettuce to detect PCR-based codominant microsatellite markers. Fifty-eight SAMPLs were identified and placed on the genetic map. Seventeen were codominant. SAMPLs were dispersed with RFLP markers on 11 of the 12 main linkage groups in lettuce, indicating that they have a similar genomic distribution. Some but not all fragments amplified by SAMPL analysis were confirmed to contain microsatellite sequences by Southern hybridization. Forty-five cultivars of lettuce and five wild species of Lactuca were analyzed to determine the allelic diversity for codominant SAMPLs. From 3 to 11 putative alleles were found for each SAMPL; 2–6 alleles were found within Lactuca sativa and 1–3 alleles were found among the crisphead genotypes, the most genetically homogeneous plant type of L. sativa. This allelic diversity is greater than that found for RFLP markers. Numerous new alleles were observed in the wild species; however, there were frequent null alleles. Therefore, SAMPL analysis is more applicable to intraspecific than to interspecific comparisons. A phenetic analysis based on SAMPLs resulted in a dendrogram similar to those based on RFLP and AFLP markers.Key words: microsatellite, Lactuca sativa, genetic variation, molecular marker.
Chromosome pairing and chiasma formation in autopolyploids of different Lathyrus species
Authors: Khawaja H. I. T. , Sybenga J. , Ellis J. R.
Published: May 2008

Chromosome pairing and chiasma formation were studied in natural and induced tetraploids (2n = 28) of Lathyrus odoratus (induced), Lathyrus pratensis (natural and induced), Lathyrus sativus (induced), and Lathyrus venosus (natural), as well as in triploids of L. pratensis and diploids of L. odoratus, L. pratensis, and L. sativus. All natural tetraploids appeared to be autotetraploids and their meiotic metaphase I behaviour was very similar to that of the induced autotetraploids, with average numbers of pairing partner switches exceeding 4 or even 5. Multivalent frequencies were high, but the numbers of chiasmata were not much higher than necessary to maintain the configurations. Interstitial chiasmata were common, but not predominant. Fertility was reduced, but sufficient for predominantly vegetatively reproducing species. The triploids of L. pratensis had an even higher multivalent frequency than the tetraploids, but still produced some viable progeny at or close to the tetraploid level, suggesting that in mixed populations of diploids and tetraploids, triploids can contribute to gene flow between the ploidy levels. There was no significant correlation between chiasma frequency and ring bivalent frequency in the diploids and multivalent frequency in the corresponding tetraploids. In the tetraploids, chiasma frequency and multivalent frequency were negatively correlated.Key words: Lathyrus, natural, induced, autotetraploid, triploid, meiosis.
Cytogenetic analysis of a self-fertilizing fish, Rivulus marmoratus: remarkable chromosomal constancy over a vast geographic range
Authors: Sola L. , Marzovillo M. , Rossi A. R. , Gornung E. , Bressanello S. , Turner B. J.
Published: May 2008

The aplocheiloid killifish Rivulus marmoratus is the only known self-fertilizing hermaphroditic vertebrate. Most natural populations consist almost entirely of hermaphrodites and comprise arrays of homozygous clones. However, in almost all populations thus far studied, clonal variation, as detected with molecular techniques, is very high. A karyological survey was carried out on specimens from Brazil, the Bahamas, Belize, and Florida (4 locales) by C-banding, silver staining, and fluorescent staining. The chromosome complement of R. marmoratus is surprisingly uniform over its vast geographic range, in terms of both chromosome number and morphology, heterochromatin distribution and composition, and nucleolar organizer region (NOR) distribution. The short arms of chromosome pair 15, where NORs are located, showed the only variation detected in this study: those of pattern A were consistently shorter than those of pattern B; moreover, the latter show positive heteropycnosis with Giemsa staining. The present data demonstrate that chromosomal variation is not a significant part of the clonal divergence in this species, even though its breeding system, by forming homozygotes for new rearrangements almost immediately, should make that variation easier to detect. The high chromosomal homogeneity is discussed in the light of the peculiar natural history of the species.Key words: hermaphrodite killifish, chromosomes, NORs, heterochromatin.
A minichromosome carrying a pigmentation gene and brook trout DNA sequences in transgenic rainbow trout
Authors: Peek Andrew S. , Wheeler Paul A. , Ostberg Carl O. , Thorgaard Gary H.
Published: May 2008

We describe the transmission of an introduced minichromosome of brook trout (Salvelinus fontinalis) origin, carrying a pigmentation gene, through three generations in rainbow trout (Oncorhynchus mykiss). The minichromosome was originally introduced into gynogenetic albino rainbow trout using γ-irradiated brook trout sperm. In the third generation, the presence of the minichromosome was correlated with pigmentation. A brook trout specific interspersed repeat DNA sequence, Fok I, was also correlated with pigmentation in these individuals. This system, the first clearly documented example of induced chromosome mediated gene transfer at the organismal level, could have applications in studies of gene mapping, development, gene regulation, and chromosome function.Key words: gynogenesis, transgenic, trout, minichromosome, Oncorhynchus mykiss.
Development and characterization of SCAR markers linked to the citrus tristeza virus resistance gene from Poncirus trifoliata
Authors: Deng Zhanao , Xiao Shunyuan , Huang Shu , Gmitter Jr. Frederick G.
Published: May 2008

Twelve new dominant randomly amplified polymorphic DNA (RAPD) fragments associated with a single dominant gene for resistance to citrus tristeza virus (CTV) were identified using bulked segregant analysis of an intergeneric backcross family. These and eight previously reported RAPDs were mapped in the resistance gene (Ctv) region; the resulting localized linkage map spans about 32 cM, with nine close flanking markers within 2.5 cM of Ctv. Seven of 20 RAPD fragments linked with the resistance gene were cloned and sequenced, and their sequences were used to design longer primers to develop sequence characterized amplified region (SCAR) markers that can be utilized reliably in marker-assisted selection, high-resolution mapping, and map-based cloning of the resistance gene. All seven cloned RAPDs were converted successfully into SCARs by redesigning primers, optimizing PCR parameters (especially the annealing temperature), or digesting amplification products with restriction enzymes. Four of the seven remained dominant markers, displaying presence–absence polymorphism patterns; the other three detected restriction site changes or length variations and thus were transformed into codominant markers. Two genomic regions rich in variability were also detected by two codominant SCAR markers.Key words: RAPD, gene mapping, citrus tristeza virus resistance.
Meiotic behavior and DNA content in alloplasmic lines of maize
Authors: Poggio Lidia , Rosato Marcela , Naranjo Carlos A.
Published: May 2008

The cytoplasm of Zea mays ssp. mexicana (teosinte) affects several inherited traits when combined with genotypes of maize (Zea mays ssp. mays). The meiotic behavior and the total DNA content of four lines of maize with teosinte cytoplasm were compared with those of the parental lines. The results obtained suggest that the cytoplasm of teosinte promotes an increase in total nuclear DNA content, perhaps through an increase of highly repetitive DNA in the knob zones. The analysis of meiotic behavior indicates that the cytoplasm of teosinte can alter the spatial distribution of the genomes, since two groups of five bivalents each were observed at a high frequency. During prophase I – anaphase I, each group of five bivalents behaves in a slightly asynchronous way with respect to the other group and, moreover, two nucleoli were observed in 10% of the cells. These results suggest that the cytoplasm of teosinte could induce changes affecting genomic structure and function in some maize genotypes. These changes are of potential importance for breeding programs and evolutionary studies.Key words: maize, teosinte, Zea mays ssp. mexicana, alloplasmy, nuclear-cytoplasmic interaction, meiotic behavior, DNA content.
A family of centromeric satellite DNAs from the European brown frog Rana graeca italica
Authors: Cardone Daniela Ester , Marotta Marcello , Rosati Claudia , Chinali Gianni , Feliciello Isidoro
Published: May 2008

Digestion of Rana graeca italica DNA with Asp718I produces highly repetitive fragments of 281 and 385 bp that were cloned and sequenced. The shorter fragment corresponds to the unit repeat (RgiS1b) of a satellite DNA. The longer fragment was found to be part of a 494-bp repeat of another satellite DNA (RgiS1a) that was cloned intact as an EcoRV fragment. RgiS1b is 97% homologous to RgiS1a, from which it seems to be derived by a single deletion. Among all species tested, only the related brown frog Rana dalmatina contained homologous repetitive DNA. The overall number of RgiS1a and RgiS1b repeats per R. graeca italica haploid genome was estimated to be 2.7 × 105. RgiS1a and RgiS1b repeats are organized in separate arrays, but repetitive units formed by various combinations of the two repeats were also observed on Southern blots. The amount of these extra repeats varies greatly among animals from the same population, representing a rare case of individual variability in the satellite DNA organization. FISH with probes specific for both satellites, or for RgiS1a only, labeled the centromeric and pericentromeric heterochromatin of all chromosomes. This indicated that RgiS1a and RgiS1b are interspersed within the same heterochromatic regions of the chromosomes.Key words: satellite DNA, nucleotide sequence analysis, tandem repeats organization, amphibian chromosomes.
Colocalization of antisense RNAs and ribozymes with their target mRNAs
Authors: Arndt Greg M. , Rank Gerry H.
Published: May 2008

The use of complementary RNA sequences such as antisense RNAs and ribozymes to regulate the expression of specific genes in eukaryotic cells has been well-documented, particularly with their application to both human gene therapy and plant biotechnology. Despite the simplicity of this approach, this technique usually results in only partial suppression of gene expression and, in some instances, even fails to regulate the gene of interest. The variation observed with antisense RNA and ribozyme-mediated regulation is further complicated by the many factors with the potential to impact on the effectiveness of these RNAs. Recent advances in the understanding of the global architecture of the nucleus, chromatin structure, and RNA metabolism provide useful and necessary information for designing novel approaches to improving antisense RNA and ribozyme regulation. These studies predict that the position of genes within the nucleus is not random and that transcripts produced from these genes follow specific tracks in migrating to the cell cytoplasm. These observations have the potential to impact significantly on the ways in which RNA-mediated forms of gene regulation are applied. The purpose of this review is to discuss the concept of colocalizing antisense RNAs and ribozymes with their target mRNAs and to introduce a variety of approaches aimed at achieving this goal.Key words: antisense RNA, ribozymes, colocalization, location effect.
Variation of nuclear ribosomal RNA genes inEragrostis tef(Zucc.) Trotter
Authors: Pillay Michael
Published: May 2008

Variation in the ribosomal RNA genes (rDNA) was examined to assess the genetic variability among 314 plants representing 28 accessions of Eragrostis tef, an important food crop. A restriction site map was constructed for the species by localization of the BamHI, BglII, DraI, EcoRI, EcoRV, NdeI, SacI, SpeI, XbaI, and XhoI sites. A comparison of this map with those of other grasses showed conservation of sites, especially in the coding region. However, a unique EcoRI site combined with a BamHI site in the 18S region may be of diagnostic value for the species. A BamHI fragment that spans the intergenic spacer was used as an indicator of length variation of rDNA repeat units. rDNA repeat units in E. tef ranged in size from 8.4 to 11.07 kbp. Considerable size variation of rDNA repeats was present among accessions, between individual plants within some accessions, and within single plants. A total of 19 spacer length (sl) phenotypes was observed in 16 accessions in which 11–42 plants were analyzed. A single restriction site polymorphism was detected in PI442115 that was also distinguished by having a single sl variant. Variation in the rRNA genes is a useful indicator of genetic diversity in E. tef germplasm.Key words: Eragrostis tef, ribosomal DNA, restriction map, genetic variation.
Segregation analysis of RFLP markers reveals a tetrasomic inheritance in apomicticPaspalum simplex
Authors: Pupilli F. , Caceres M. E. , Arcioni S. , Quarin C. L.
Published: May 2008

Apomictic tetraploid Paspalum simplex was crossed with colchicine-doubled diploid sexual plants belonging to the same species. Homologous genomic probes were selected from a partial PstI genomic library for their capacity to detect alleles specific to the apomictic parent, and their segregation was analyzed in the F1progeny. High levels of polymorphism between apomictic and sexual genotypes were recorded. The heterozygosity was high in both tetraploid and diploid genotypes but the differences between them were not as great as expected. In the sexual parent, some markers segregated as either a monoallelic duplex or a diallelic duplex, while several allelic configurations were observed in the apomictic parent. The segregation of double-dose monoallelic fragments demonstrated the tetrasomic inheritance of apomictic P. simplex. The correlations between apomixis, ploidy level, and tetrasomic inheritance are discussed.Key words: apomixis, tetrasomic inheritance, Paspalum simplex, molecular markers.
Development of molecular markers linked to a gene controlling fruit acidity in citrus
Authors: Fang D. Q. , Federici C. T. , Roose M. L.
Published: May 2008

Fruit juice pH, titratable acidity, or citric acid content was measured in 6 populations derived from an acidless pummelo (pummelo 2240) (Citrus maxima (Burm.) Merrill). The acidless trait in pummelo 2240 is controlled by a single recessive gene called acitric. Using bulked segregant analysis, three RAPD markers were identified as linked to acitric. RAPD marker OpZ20410, which mapped 1.2 cM from acitric, was cloned and sequenced, and a sequence characterized amplified region (SCAR) marker (SCZ20) was developed. The SCZ20-410 marker allele that is linked to the acitric allele occurs only in pummelo 2240 and other pummelos, and therefore, this SCAR marker should be useful as a dominant or codominant marker for introgressing acitric into mandarins and other citrus species. Using the cloned OpZ20410band as a hybridization probe revealed a codominant RFLP marker called RFZ20 that mapped 1.2 cM from acitric. Progeny homozygous (acac) for the acitric allele had citric acid content below 10 μM, the minimum level detectable by high pressure liquid chromatography. The citric acid content of fruit juice from progeny predicted to be heterozygous (Acac) for acitric by the above markers was about 30% lower than that of juice from individuals predicted to be homozygous (AcAc) for the normal acid allele. Markers OpZ20410, SCZ20, and RFZ20 were highly polymorphic among 59 citrus accessions, and using one or more of these markers would allow citrus breeders to select seedling progeny heterozygous for acitric in nearly all crosses between pummelo 2240 or its offspring and other citrus genotypes.Key words: Citrus, fruit acidity, citric acid, RAPD, SCAR, RFLP.