Genetic diversity of <Emphasis Type="Italic">Opuntia</Emphasis> spp. varieties assessed by classical marker tools (RAPD and ISSR)

Opuntia, commonly named “nopal” in Mexico, is an important crop for its agronomical, economical, ecological and cultural value. Furthermore, it is known for its taxonomic complexity. In this paper, we report the genetic variability of 52 Opuntia cultivars with agronomic and economic importance, classified into 12 different species using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. Ten primers, five for each marker type, were selected to assess their ability to detect polymorphisms in this plant accesions/varieties. Both marker systems generated a total of 307 bands, of which 50.8 % were polymorphic with an average of 15.6 polymorphic bands per primer. Thus, we assume that Mexican Opuntia varieties present broad genetic variation. Based on percentage of polymorphic bands; resolving power; polymorphic information content; and Marker Index, the K-12 (RAPD) and IS-06 (ISSR) primers were the most informative ones. Clusters obtained from RAPD, ISSR and a combination of both data sets did not match the actual taxonomic classification. On the other hand, the putative varieties currently classified in the same species were not located in the same cluster. Besides, the varieties included in O. ficus-indica, O. albicarpa and O. megacantha showed broad variation but were not well defined into separate clades; these cultivars possibly have common ancestry. The results presented here support our hypothesis about the existence of a smaller number of Opuntia species in accordance with those currently described, but with high intraspecific genetic variation.     

Development of Dof (DNA binding with one finger) transcription factor gene-specific primers through data mining as a functional marker and their use for genetic diversity study in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) germplasm

The genetic diversity among Hordeum vulgare L. species were assessed based on PCR amplification pattern derived from 75 set of Dof domain and Dof genes specific primers. Multiple bands showing variability in terms of both number and sizes of bands ranging from 0.1 to 3.0 kbp were observed. Out of a total of 2449 bands, 2328 polymorphic and 121 monomorphic bands were obtained and the percentage of polymorphism ranged from 70.27 to 100%. A very high degree of polymorphism was observed with all the primers except HvDof3, HvDof4, HvDof10, HvDof16, HvDof18, HvDof18, HvDof24, Dof4, Dof11, Dof13, Dof15, Dof16, Dof19, Dof20, Dof21, Dof22, Dof23, Dof28, dof38, sbDof23 and sbDof24 primers. Unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on Jaccard’s similarity coefficient matrix. According to results, the genetic resources and diversity in barley germplasm of H. vulgare were rich. The number of polymorphic fragments per primer detected ranged from 11 to 56 bands with an average of 32.65 bands. Average polymorphic information content (PIC) was 0.81 in overall Dof domain and gene specific primers. HvDof 39 showed the highest PIC (0.99) which can be a good candidate primer to verify genetic diversity in H. vulgare. The unweighted pair-group method of the arithmetic average and principal coordinate analysis showed a clear distinction among the genotypes and the genotypes divided into three clusters in the dendrogram results. A model-based structure analysis revealed the presence of three groups. The study showed that genetic variation and population structure are determined among the species of H. vulgare collected from different geographical origins.     

Differentiation of petroleum hydrocarbon-degrading <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. based on PCR-RFLP of the 16S-23S rDNA intergenic spacer region

Genetic diversity among 43 petroleum hydrocarbon-degrading Pseudomonas belonging to four different species and the type strain Pseudomonas aeruginosa MTCC1034 was assessed by using restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified 16S–23S rDNA intergenic spacer regions (ISRs) polymorphism. PCR amplification from all Pseudomonas species yielded almost identical ISR amplicons of “?” 800 bp and in nested PCR of “?” 550 bp. The RFLP analysis with MboI and AluI revealed considerable intraspecific variations within the Pseudomonas species. The dendrogram constructed on the basis of the PCR-RFLP patterns of 16S–23S rDNA intergenic spacer regions differentiated all the species into seven different clusters.     

Evaluation of genetic homogeneity of in vitro-raised plants of <Emphasis Type="Italic">Tecomella undulata</Emphasis> (Sm.) Seem. using molecular markers

Tecomella undulata (Sm.) Seem (family Bignoniaceae) is an economically and pharmaceutically important timber tree of arid regions of India. Overexploitation of natural stands coupled with minimal conservation and reforestation efforts has led to its incorporation in list of endangered species. This monotypic genus can be propagated only through seeds as no methods are available for its vegetative propagation. Therefore, protocol for multiplication of T. undulata via direct regeneration using nodal segments from mature trees has been standardized. Authentication of genetic homogeneity of these in vitro-raised plants is necessary for commercial-scale application of the developed micropropagation protocol. PCR-based molecular markers which have emerged as simple, fast, reliable, and labor-effective tools for testing the genetic homogeneity of in vitro-raised plants were used in the present study. Arbitrary (random amplified polymorphic DNA, RAPD), semi-arbitrary (inter-simple sequence repeat, ISSR; start codon targeted (SCoT) polymorphism), and sequence-based (simple sequence repeat, SSR) markers were used. DNA samples of shoots maintained in vitro for 2 years collected after every 4 subculture cycles (of 3 weeks each) and field-transferred plantlets were compared with the mother tree DNA using 131 primers (25 each of RAPD, ISSR, SCoT and 56 SSR). Scorable unambiguous and reproducible DNA fragments were produced by 77 (21 RAPD, 20 ISSR, 22 SCoT and 14 SSR) primers. A total of 71, 93, 94, and 42 distinct and scorable DNA fragments were produced by RAPD, ISSR, SCoT, and SSR primers respectively with an average of 3.38, 4.65, 4.27, and 3.0 DNA fragments per primer. The true-to-type nature of the in vitro-raised plants of T. undulata undergoing up to 32 subculture passages over a period of approximately 2 years was authenticated by monomorphic DNA fragments amplified with all primer combinations. Therefore, the developed micropropagation protocol can be safely used on a commercial scale for multiplying T. undulata plants.     

Cytotype-specific ISSR profiles and karyotypes in the Neotropical genus <Emphasis Type="Italic">Eigenmannia</Emphasis> (Teleostei: Gymnotiformes)

The genus Eigenmannia (Teleostei: Gymnotiformes), a widely distributed fish genus from the Neotropical region, presents very complex morphological patterns and many taxonomic problems. It is suggested that this genus harbors a species complex that is hard to differentiate using only morphological characteristics. As a result, many species of Eigenmannia may be currently gathered under a common name. With the objective of providing new tools for species characterization in this group, an analysis of the polymorphism of DNA inter-simple sequence repeats (ISSR), obtained by single primer amplification reaction (SPAR), combined with karyotype identification, was carried out in specimens sampled from populations of the Upper Paraná, São Francisco and Amazon river basins (Brazil). Specific ISSR patterns generated by primers (AAGC)₄ and (GGAC)₄ were found to characterize the ten cytotypes analyzed, even though the cytotypes 2n = 38 and 2n = 38 XX:XY, from the Upper Paraná basin, share some ISSR amplification patterns. The geographical distribution of all Eigenmannia specimens sampled was inferred, showing the cytotype 2n = 31/2n = 32 as the most frequent and largely distributed in the Upper Paraná basin. The cytotype 2n = 34 was reported for the first time in the genus Eigenmania, restricted to the São Francisco basin. Polymorphic ISSR patterns were also detected for each cytotype. Considering our results and the data reported previously in the literature, it is suggested that many of the forms of Eigenmannia herein analyzed might be regarded as different species. This work reinforces the importance of employing diverse approaches, such as molecular and cytogenetic characterization, to address taxonomic and evolutionary issues.     

Differentiation of <Emphasis Type="Italic">Vaccinium</Emphasis> Cultivars and Wild Clones Using RAPD Markers

The potential use of the random amplified polymorphic DNA (RAPD) technique for characterization and assessment ofgenetic relatedness was investigated in 13 wild cranberry (Vaccinium macrocarpon Ait) clones collected from Newfoundlandand Labrador, Canada. RAPD markers were also used to distinguish representatives of three different Vaccinium species.Twenty-two decamer arbitrary primers yielded informative, reproducible and polymorphic banding patterns in 13 cranberryclones and produced a total of 134 bands, of which 114 were polymorphic. The UPGMA cluster analysis separated the clonesinto two main groups: Cluster I of seven and Cluster II of six with 0.62 to 0.91 and 0.50 to 0.77 Nei and Li’s similarity range,respectively. In another experiment, a subset of eight primers was used and the RAPD markers discriminated the threeVaccinium species: cranberry, lowbush blueberry (V. angustifolium Ait) and lingonberry (V. vitis-idaea L). The RAPDpolymorphisms will be useful for Vaccinium genotype differentiation and the technology should be valuable for themaintenance of germplasm banks and the efficient choice of parents in the current Vaccinium germplasm improvementprogram.     

Molecular diversity and genetic relationships in <Emphasis Type="Italic">Secale</Emphasis>

The objective of this study was to quantify the molecular diversity and to determine the genetic relationships among Secale spp. and among cultivars of Secale cereale using RAPDs, ISSRs and sequence analysis of six exons of ScMATE1 gene. Thirteen ryes (cultivated and wild) were genotyped using 21 RAPD and 16 ISSR primers. A total of 435 markers (242 RAPDs and 193 ISSRs) were obtained, with 293 being polymorphic (146 RAPDs and 147 ISSRs). Two RAPD and nine ISSR primers generated more than 80% of polymorphism. The ISSR markers were more polymorphic and informative than RAPDs. Further, 69% of the ISSR primers selected achieved at least 70% of DNA polymorphism. The study of six exons of the ScMATE1 gene also demonstrated a high genetic variability that subsists in Secale genus. One difference observed in exon 1 sequences from S. vavilovii seems to be correlated with Al sensitivity in this species. The genetic relationships obtained using RAPDs, ISSRs and exons of ScMATE1 gene were similar. S. ancestrale, S. kuprijanovii and S. cereale were grouped in the same cluster and S. segetale was in another cluster. S. vavilovii showed evidences of not being clearly an isolate species and having great intraspecific differences.     

First microsatellite markers for <Emphasis Type="Italic">Paspalum plicatulum</Emphasis> (Poaceae) characterization and cross-amplification in different <Emphasis Type="Italic">Paspalum</Emphasis> species of the Plicatula group

Background

Paspalum plicatulum is a perennial rhizomatous grass with natural diploid and polyploid cytotypes. It is a member of Plicatula, which has historically been recognized as a highly complex group containing species of ecological, ornamental and forage importance. The complex nature of the P. plicatulum genome makes it a challenging species for genetic research. This study aimed to develop and characterize microsatellite molecular markers in P. plicatulum and to evaluate their transferability to other Plicatula group species.

Findings

Microsatellite sequences were identified from three enriched libraries from P. plicatulum. Specific primers were designed, and 25 displayed polymorphism when screened across 48 polyploid Paspalum spp. genotypes. The number of bands per locus ranged from 2 to 17, with a mean of 8.65. Private bands for each species were identified; the highest number of private bands was observed for P. plicatulum in 52% of the loci analyzed. The mean polymorphism information content of all loci was 0.69, and the mean discriminatory power was 0.82. Microsatellite markers were satisfactorily cross-amplified for the eight tested Plicatula-group Paspalum species, with P. atratum exhibiting the highest transferability rate (89.86%). STRUCTURE and Discriminant Analysis of Principal Components separated accessions into three groups but did not reveal separation of the accessions according to species.

Conclusions

This study describes the first microsatellite markers in P. plicatulum, which are polymorphic, efficient for the detection and quantification of genetic variation, and show high transferability into other species of the Plicatula group. This set of markers can be used in future genetic and molecular studies necessary for the proper development of conservation and breeding programs. Private bands within the markers can be used to assist in species identification.

     

Evaluation of genetic diversity of <Emphasis Type="Italic">Clinacanthus nutans</Emphasis> (Acanthaceaea) using RAPD,ISSR and RAMP markers

Three polymerase chain reaction (PCR) techniques were compared to analyse the genetic diversity of Clinacanthus nutans eight populations in the northern region of Peninsular Malaysia. The PCR techniques were random amplified polymorphic deoxyribonucleic acids (RAPD), inter-simple sequence repeats (ISSR) and random amplified microsatellite polymorphisms (RAMP). Leaf genomic DNA was PCR amplified using 17 RAPD, 8 ISSR and 136 RAMP primers . However, only 10 RAPD primers, 5 ISSR primers and 37 RAMP primers produced reproducible bands. The results were evaluated for polymorphic information content (PIC), marker index (MI) and resolving power (RP). The RAMP marker was the most useful marker compared to RAPD and ISSR markers because it showed the highest average value of PIC (0.25), MI (11.36) and RP (2.86). The genetic diversity showed a high percentage of polymorphism at the species level compared to the population level. Furthermore, analysis of molecular variance revealed that the genetic diversity was higher within populations, as compared to among populations of C. nutans. From the results, the RAMP technique was recommended for the analysis of genetic diversity of C. nutans.     

Genetic diversity of the African poplar (<Emphasis Type="Italic">Populus ilicifolia</Emphasis>) populations in Kenya

We evaluated the genetic diversity of the African poplar (Populus ilicifolia) populations found in Kenya compared with reference samples of five poplar species from North America and one species introduced in Kenya from India (KEFRI-Kenya). Amplified fragment length polymorphism (AFLP) was used with the objective of providing important information for breeding and in situ/ex situ conservation of this species. Samples collected from three locations along the species’ natural range (Athi, Ewaso Nyiro, and Tana rivers) were compared with four samples of locally planted Populus deltoides stand introduced from India and ten reference samples from North America. Six AFLP primer combinations produced 521 clear bands for analysis. The percentage polymorphic loci were lowest in Tana (20.4 %) and highest in Athi (40.6 %). The average heterozygosity across the studied populations was between 0.07 and 0.3. AMOVA revealed more genetic variation partitioning within population (87 %; P?<?0.01) than among populations (13 %; P?<?0.01) suggesting significant genetic variation between populations. Further, UPGMA delineation showed two clusters of the Tana, Athi, and Ewaso Nyiro populations clustered together compared to the North America and India/KEFRI reference samples. Moreover, the study showed that the Athi population is more diverse than those of Tana and Ewaso Nyiro and may be important for conservation, domestication, and improvement studies. The genetic differentiation (F _ST ?=?0.134) among Kenyan P. ilicifolia populations suggests limited possibility of gene flow between these populations.     

Variation and Divergence of Multilocus Genome Markers in the Species of the Genus <Emphasis Type="Italic">Chironomus</Emphasis> (Diptera,Chironomidae)

Variation and divergence patterns of the multilocus genome markers in twelve Chironomus species belonging to the plumosus and piger sibling-species groups were examined by use of polymerase chain reaction with arbitrary primers (RAPD method). The chironomid species showed high levels of the RAPD markers polymorphism. The genetic distances (GD) were determined between the species within the group of closely related species, as well as between the species from different groups. The estimates obtained characterized the divergence levels between the sibling species (GD = 0.739) and morphologically distinct species (GD = 0.935). Comparison of the variation and divergence levels of the RAPD markers with those for the other genome markers, namely, the enzyme-coding genes and chromosomes (gene linkage groups) have demonstrated different divergence rates for different genome components during speciation of chironimids.     

Real-time PCR assay for rapid,efficient and accurate detection of <Emphasis Type="Italic">Paramyrothecium roridum</Emphasis> a leaf spot pathogen of <Emphasis Type="Italic">Gossypium</Emphasis> species

Here we report a highly sensitive real-time PCR (qPCR) assay to detect Paramyrothecium roridum from pure culture and infected samples of cotton plants. A specific set of primer pair pMyro F/R is designed to target the 185 bp ITS region of rDNA of Paramyrothecium roridum species and validated using qPCR. The fluorescence signals were detected above the baseline threshold from samples containing Paramyrothecium roridum DNA, whereas other samples did not produce any fluorescence or produced fluorescence which did not reach detection threshold values. A single dissociation peak of increased fluorescence was obtained for the specific primers at 92.2 °C melting temperature. The limit of detection using SYBR Green dye in this assay was up to 0.1 pg per µL of DNA from pure culture of P. roridum. The assay is accurate, sensitive, less laborious and time saving for detection of P. roridum in infected tissues of cotton.     

Characterization of the complete mitochondrial genome of flower-breeding <Emphasis Type="Italic">Drosophila incompta</Emphasis> (Diptera,Drosophilidae)

Drosophila incompta belongs to the flavopilosa group of Drosophila, and has a restricted ecology, being adapted to flowers of Cestrum as feeding and oviposition sites. We sequenced, assembled, and characterized the complete mitochondrial genome (mtDNA) of D. incompta. In addition, we performed phylogenomic and polymorphism analyses to assess evolutionary diversification of this species. Our results suggest that this genome is syntenic with the other published mtDNA of Drosophila. This molecule contains 15,641 bp and encompasses two rRNA, 22 tRNA and 13 protein-coding genes. Regarding nucleotide composition, we found a high A?T bias (76.6 %). The recovered phylogenies indicate D. incompta in the virilis–repleta radiation, as sister to the virilis or repleta groups. The most interesting result is the high degree of polymorphism found throughout the D. incompta mitogenome, revealing pronounced intrapopulational variation. Furthermore, intraspecific nucleotide diversity levels varied between different regions of the genome, thus allowing the use of different mitochondrial molecular markers for analysis of population structure of this species.     

Analysis of genetic diversity and population structure in <Emphasis Type="Italic">Capsicum</Emphasis> landraces from North Eastern India using TE-AFLP markers

North eastern (NE) India harbours a precious germplasm repository of Capsicum in the form of various landraces. The present study was undertaken to characterise the extent of genetic variation present in different Capsicum landraces from north eastern India. A set of 171 Capsicum accessions were characterised using three-endonuclease amplified fragment length polymorphism (AFLP) markers. Out of 416 bands obtained from six primer combinations, 254 (61 %) were polymorphic. The pairwise genetic dissimilarity among accessions ranged from 0.03 to 0.97. Cluster analysis based on neighbour joining showed two major clusters. Cluster I contained most of the bhut jolokia accessions whereas cluster II contained all of the Capsicum annuum genotypes. Similar grouping was observed with population STRUCTURE analysis as well as principle coordinate analysis. Analysis of molecular variance (AMOVA) revealed 45 and 54 % variation among and within populations, respectively. This information on population structure analysis and molecular characterisation will be helpful for effective utilisation of this germplasm in Capsicum improvement programs.     

Molecular evidence for a natural diploid hybrid between <Emphasis Type="Italic">Miscanthus</Emphasis><Emphasis Type="Italic">sinensis</Emphasis> (Poaceae) and <Emphasis Type="Italic">M. sacchariflorus</Emphasis>

The hybrid origin of Miscanthus purpurascens has previously been proposed, primarily because of its intermediate morphology. In this study, phylogenies based on the DNA sequences from the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA ITS), on the DNA sequences of the trnL intron and trnL-F intergenic spacer of chloroplast DNA, and on amplified fragment length polymorphism (AFLP) fingerprinting confirm that M. purpurascens originated through homoploid hybridization between M. sinensis and M. sacchariflorus. Two different types of ITS sequences were identified from almost all plants of M. purpurascens. One type was found to be closely related to M. sinensis and the other to M. sacchariflorus. Miscanthus purpurascens was found to possess many M. sinensis- and M. sacchariflorus-specific AFLP bands but no band specific to itself. Clustering with the Unweighted Pair Group Method with Arithmetic Mean and principal coordinate analysis based on the AFLP data also demonstrated that M. purpurascens is an approximate intermediate of the two species. In addition, M. purpurascens has the plastid genome of M. sinensis or M. sacchariflorus, suggesting that either species could be its maternal parent. All specimens of M. purpurascens and its coexisting parental species are identified as diploids (2n = 2x = 38). Possible mechanisms of natural hybridization, hybrid status, chloroplast DNA recombination, and evolutionary implications of this hybridization are also discussed.     

Maternal care,larviparous and oviparous reproduction of <Emphasis Type="Italic">Hypoaspis larvicolus</Emphasis> (Acari: Laelapidae) feeding on astigmatid mites

Hypoaspis larvicolus (Acari: Laelapidae) (first report from Turkey) occurred together with Sancassania polyphyllae (Acari: Acaridae) on the larvae of the scarab beetle, Polyphylla fullo (Coleoptera: Scarabaeidae), that were feeding on the roots of strawberry in Aydin, Turkey. Laboratory studies were conducted to (1) observe whether H. larvicolus feeds and completes its life cycle on the various stages of S. polyphyllae or other astigmatid mites, such as Acarus siro, Carpoglyphus lactis and Tyrophagus putrescentiae (Acaridae), and to determine its population growth when feeding on these prey, and (2) to determine development periods, longevity and fecundity of H. larvicolus feeding on C. lactis. Hypoaspis larvicolus females did not feed on S. polyphyllae, but fed, developed and reproduced when A. siro, C. lactis or T. putrescentiae were provided as prey. Hypoaspis larvicolus is larviparous as well as oviparous. The female lays eggs or gives birth to larvae. If a female gives birth to a larva, it is attached under the female’s venter for 1–2 days, a phenomenon recorded for the first time in Hypoaspis; in fact, for the first time in mites. The results of the population growth experiments revealed that H. larvicolus feeding on C. lactis produced the highest number of eggs, juveniles and adults. The developmental periods of H. larvicolus feeding on C. lactis at life-cycle path I (larva to adult) and II (egg to adult) were 12.2?±?0.3 and 15.6?±?0.6 days (females) and 19.5?±?0.2 and 20.9?±?0.4 days (males), respectively. Longevity of females versus males of H. larvicolus was 120.6?±?7.2 versus 91.6?±?13.1 days (life cycle I) and 110.0?±?27.7 versus 118.3?±?10.9 days (life cycle II), respectively.     

Diversity,molecular phylogeny and fingerprint profiles of airborne <Emphasis Type="Italic">Aspergillus</Emphasis> species using random amplified polymorphic DNA

In the present study, diversity and phylogenetic relationship of Aspergillus species isolated from Tehran air was studied using random amplified polymorphic DNA (RAPD)–polymerase chain reaction (RAPD-PCR). Thirty-eight Aspergillus isolates belonging to 12 species i.e. A. niger (28.94 %, 11 isolates), A. flavus (18.42 %, 7 isolates), A. tubingensis (13.15 %, 5 isolates), A. japonicus (10.52 %, 4 isolates), A. ochraceus (10.52 %, 4 isolates), and 2.63 %, 1 isolate from each A. nidulans, A. amstelodami, A. oryzae, A. terreus, A. versicolor, A. flavipes and A. fumigatus were obtained by settle plate method which they were distributed in 18 out of 22 sampling sites examined. Fungal DNA was extracted from cultured mycelia of all Aspergillus isolates on Sabouraud Dextrose Agar and used for amplification of gene fragments in RAPD-PCR using 11 primers. RAPD-PCR data was analyzed using UPGMA software. Resulting dendrogram of combined selected primers including PM1, OPW-04, OPW-05, P160, P54, P10 and OPA14 indicated the distribution of 12 Aspergillus species in 8 major clusters. The similarity coefficient of all 38 Aspergillus isolates ranged from 0.02 to 0.40 indicating a wide degree of similarities and differences within and between species. Taken together, our results showed that various Aspergillus species including some important human pathogenic ones exist in the outdoor air of Tehran by different extents in distribution and diversity and suggested inter- and intra-species genetic diversity among Aspergillus species by RAPD-PCR as a rapid, sensitive and reproducible method.     

Development of intron targeted amplified polymorphic markers of metal homeostasis genes for monitoring their introgression from <Emphasis Type="Italic">Aegilops</Emphasis> species to wheat

The identification of transfers of useful alien genes for metal homeostasis from non-progenitor Aegilops species using the widely available anchored wheat SSR markers is difficult due to their lower polymorphism with the distant related wild species and the lack of locus specificity further restricts their application. The present study deals with the development of intron targeted amplified polymorphic (ITAP) markers for the metal homeostasis genes present on chromosomes of groups 2 and 7 of Triticeae. The mRNA sequences of 27 metal homeostasis genes were retrieved from different plant species using NCBI database and their BLASTn was performed against the wheat draft genome sequences in Ensemblplants to get exonic and intronic sequences of the corresponding metal homeostasis genes in wheat. The ITAP primers were developed in such a way that they would anneal to the conserved flanking exonic regions of the genes and amplify across highly variable introns within the PCR limits. The primers led to the amplification of variable intronic sequences of genes with polymorphism between non-progenitor Aegilops species and the recipient wheat cultivars. Further, the polymorphic ITAP markers were used to characterize the transfers of metal homeostasis genes from the non-progenitor Aegilops species to the BC₂F₅ wheat-Aegilops derivatives, developed through induced homoeologous pairing. The derivatives with significant percent increase in grain Fe and Zn content over the elite cultivar PBW343 LrP showed the introgression of some of the useful Aegilops alleles of the metal homeostasis genes. The use of different metal homeostasis genes using this approach is the first report of the direct contribution of the genes for increasing the grain micronutrient content for developing biofortified wheat lines with reduced linkage drag.     

Evaluation of the Use of Selective PCR Amplification of LPS Biosynthesis Genes for Molecular Typing of <Emphasis Type="Italic">Leptospira</Emphasis> at the Serovar Level

Leptospirosis is an important epidemic zoonosis worldwide. Currently, there are more than 250 Leptospira pathogenic serovars known that can potentially infect humans. Conventional classification of leptospires with the serovar as the basic taxon, based on serological recognition of lipopolysaccharide (LPS) composition does not correlate well with species determination, based on general genomic features. Here, we investigate the selective amplification of polymorphic regions from the LPS biosynthesis loci (rfb) as a potential tool for serovar typing of Leptospira interrogans species. Eight pairs of primers were designed to target six ORFs from the rfb operon with varying levels of sequence polymorphism. They were tested both separately and multiplexed. Half of these primer pairs produced serovar-specific amplicons, allowing the identification of some specific serovars and also groups of serovars. It was shown that the serovar classification of Leptospira can be accessed by selective amplification of rfb operons in some cases, which may permit a parallel between the serological and the genomic classifications of Leptospira. As a conclusion, the selective amplification of rfb generated promising and already useful results, but it appears necessary to characterize a larger variety of Leptospira genomes or rfb operons to fully develop this method.     

Analysis of DNA polymorphism in a relict Uralian species,large-flowered foxglove (<Emphasis Type="Italic">Digitalis grandiflora</Emphasis> Mill.), using RAPD and ISSR markers

Genetic polymorphism of the Uralian relict plant species, large-flowered foxglove Digitalis grandiflora Mill. (family Scrophulariaceae), was examined using RAPD and ISSR techniques. A total of 149 RAPD and 74ISSR markers were tested. The indices characterizing polymorphism and genetic diversity were calculated. The data obtained pointed to a high level of genetic variation of D. grandiflora (P ₉₅ = 65%). The cenopopulation examined was weakly differentiated with most of genetic diversity accounted by within-population differentiation.