Academic literature on the topic 'Secondary digest-amplified fragment length polymorphism'

The objective of this study was to evaluate the genetic relationships of 28 chickpea accessions from diverse origin using AFLP markers. On average, 13 polymorphic bands per primer were observed in AFLP analysis. The average polymorphic information content (PIC) was 0.71, ranging from 0.48 to 0.92. The lowest and the highest PIC value were recorded for primer P-GAG/M-GC and P-AT/M-GC, respectively. The average GD, based on Fst values among the 21 accessions was 0.42, ranging from 0.61 to 0.16. From the UPGMA dendrogram, it is discernible that material taken for the analysis can be divided in four clusters. The results indicate that the greatest genetic diversity occurs in Afghanistan, Iran and Lebanon. In many cases, the diversity between individuals of an accession is as great as between individuals of different accessions. Based on DNA markers it is concluded that there are three centers of diversity for chickpea: Pakistan-Afghanistan, Iran-Turkey and Syria-Lebanon. India and Ethiopia, which were previously considered as a secondary center of diversity for chickpea, showed lower diversity than the above regions.

ABSTRACT Genetic diversity among Sodalis glossinidius populations was investigated using amplified fragment length polymorphism markers. Strains collected from Glossina palpalis gambiensis and Glossina morsitans morsitans flies group into separate clusters, being differentially structured. This differential structuring may reflect different host-related selection pressures and may be related to the different vector competences of Glossina spp.

Several divergent sympatry mtDNA lineages have been described in redlip mullet Liza haematocheilus, and this high inter-lineage divergence raises questions about the taxonomic status of L. haematocheilus lineages in the north-western Pacific. In this study, the amplified fragment length polymorphism technique was employed to examine genetic structure of L. haematocheilus and estimate the level of independence of the different mtDNA lineages in the north-western Pacific. A total of 186 bands were amplified from 91 individuals among 8 populations by 4 primer combinations and the percentage of polymorphic bands was 91.74%. The Unweighted Pair Group Method with Arithmetic Mean tree based on Nei genetic distance revealed two clusters (North Clade and South Clade). Molecular variance analysis and pairwise FST supported the separation of north and south populations of L. haematocheilus in the north-western Pacific. The incongruence between nuclear groups and mitochondrial lineages suggests the three distinct lineages do not represent cryptic species and the presence of divergent mitochondrial lineages in the same sample is a result of secondary contact after an extended period of isolation. The Pleistocene isolation and biological characteristics of species may be responsible for the genetic differentiation of L. haematocheilus.

AbstractMorphologically, early immature stages of the economically important pest called screwworms, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), and non-pest secondary screwworms, Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae), are nearly indistinguishable. Correct identification is crucial to the ongoing eradication and exclusion program protecting the United States, Mexico and Central America from reinvasion of screwworms persistent in South America and the Caribbean. Amplified fragment length polymorphism (AFLP) polymerase chain reaction was used to differentiate populations of C. hominivorax and to discriminate them from C. macellaria. Ten primer pairs screened for interspecific discrimination of C. hominivorax from C. macellaria showed 52 discrete bands, allowing the two species to be readily distinguished; divergent branches on resulting dendrograms showed 100% bootstrap support. C. macellaria populations grouped at the 92% level; C. hominivorax populations grouped at the 68% level. Of the 52 bands, seven were monomorphic for both species, 22 were specific to C. macellaria, ten were present only in C. hominivorax and the remaining 13 bands differentiated C. hominivorax populations. Separate studies using ten strains of C. hominivorax showed a higher level of genetic similarity within than between populations. Analyses using 72 bands (19 monomorphic bands, 53 bands grouped all ten strains at the 58% similarity level) resolved seven mutant strains from Mexico (85% similarity level); all ten strains were resolved at the 72% similarity level. Diagnostic bands were identified for species and strain identification. We conclude that AFLP can be a valuable tool for studies of interspecific and intraspecific genetic variation in screwworm populations.

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set ofH. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA ofEperythrozoon suis, Mycoplasma genitalium, andBartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI andMnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felisinfection in cats.

ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called “pseudo-T-RFs” since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.

Plum pox virus (PPV) was detected in wild apricot and cultivated plum maintained in a germ plasm collection in Kazakhstan. Both isolates were typed as D strain, with no evidence of recombination. The virus was detected by triple-antibody sandwich enzyme-linked immunosorbent assay (ELISA) utilizing the universal PPV-specific monoclonal antibody (MAb) 5B as the secondary antibody, and by reverse-transcription polymerase chain reaction (RT-PCR) assay using primers that amplified a 243-bp fragment in the C-terminus of the coat protein (CP) coding region. Immunocapture (IC) RT-PCR was used to detect PPV in nine wild apricot accessions, including eight ELISA-negative and one ELISA-positive. The plum and apricot isolates reacted positively in Western blot assay with the universal MAb 5B, and negatively with the strain-M-specific MAb-AL. Restriction fragment length polymorphism analysis applied to the amplified 243-bp fragment showed that restriction sites for AluI and RsaI were present in the were present in the plum and apricot samples. An amplified 836-bp cDNA fragment derived from the P3-6K1 coding region of both isolates had restriction profiles typical for strain D. Nucleotide identities of 99 to 100% were observed for the 243-bp fragments of the Kazakhstan isolates when compared with the corresponding regions of strain D, and 94 to 95% identity with strain M. Nucleotide sequence analysis of the entire CP coding region of the plum and apricotisolates resulted in the identification of a unique deletion of six nucleotides (two deduced proline amino acid residues) in the N-terminal region in the plum isolate. This is the first deletion of this nature observed among PPV isolates. The DAG motif was present in both isolates. Several nucleotide substitutions in the CP coding region were common to the plum and apricot isolates and appear to be unique to the Kazakstan isolates. This suggests a close relationship between the isolates.

Diverse isolates of the soilborne wilt fungi Verticillium dahliae and V. albo-atrum were studied to understand the nature and origins of those infecting cruciferous hosts. All isolates from cruciferous crops produced microsclerotia, and the majority produced long conidia with a high nuclear DNA content; these isolates were divided into two groups by amplified fragment length polymorphism (AFLP) analysis. One group could be subdivided by other criteria such as rRNA sequences and mitochondrial DNA restriction fragment length polymorphism (RFLP) analysis. Two crucifer isolates were short spored and had a low nuclear DNA content. The results are consistent with the crucifer isolates being interspecific hybrids. The long-spored isolates are best regarded as amphihaploids (or allodiploids) with the AFLP groups probably each representing separate interspecific hybridization events. The short-spored crucifer isolates appear to be derived from interspecific hybrids and are here called ‘secondary haploids’. Molecular evidence suggests that one parent in the crosses was similar to V. dahliae. The other parent of the amphihaploids seems to have been more similar to V. albo-atrum than to V. dahliae, but was distinct from all isolates of either species so far studied. The implications for the taxonomy of crucifer isolates are discussed and the use of the name V. longisporum, proposed elsewhere for just some of these isolates, is discouraged.

To reduce the level of redundancy in a collection of cultivated lettuce, data from 160 amplified fragment length polymorphism (AFLP) fragments and 10 polymorphic microsatellites were used in combination with passport data and morphological data, the latter obtained from an experimental field trial performed for verification purposes. Based on the observed distribution of the number of marker differences between and within accessions, a minimum of three AFLP differences and two microsatellite differences were regarded as levels warranting distinction between accessions in the redundancy analysis. The strategy followed in the redundancy analysis was mainly based on the confirmation of duplication by each of two independently generated data sources. The molecular data were used for the validation as well as the identification of potential duplicates, revealing a total number of 198 redundancies, corresponding to 12.9% of the total collection. Trueness to type, number of characterization and evaluation data, and collection management considerations, such as available seed quantities and germination percentages, were used as primary, secondary and tertiary criteria to decide which accession from duplication groups to maintain in the collection. Removal of accessions showed negligible effects on total collection diversity, as quantified for AFLPs and microsatellites, characterization and evaluation traits and resistance profiles against downy mildew pathotypes, indicating that the applied strategy was effective.

The complete A+T - rich region of Aedes aegypti mitochondrial DNA has been cloned and sequenced. In Argentinean populations of the species, a polymorphism in the length of the amplified fragment was observed. Nucleotide sequence comparison of the shortest and longest A+T - rich amplified fragments detected revealed the presence of 2 types of tandemly repeated blocks. The size variation observed in natural populations is mainly due to the presence of a variable number of a 181 bp tandem repeat unit, located toward the 12S rRNA gene end. The size of the longest A+T - rich region was of 2070 bp, representing the largest control sequence reported for any mosquito species. Few relevant short blocks of primary-sequence similarity conserved in the control region of mosquitoes and other insects were detected scattered throughout the whole region. Five putative stem-loop secondary structures were found, one of them flanked by conserved sequences described in other insects. Our results suggest that there are no universal models of structure–function relations in the control region of insect mtDNA. In addition, we identified a short A+T - rich variable segment in the Ae. aegyti control region that would be suitable for population genetic studies.Key words: mitochondrial DNA, A+T - rich region, repeated elements, conserved blocks, Aedes aegypti.

Protoplast fusion of Lolium perenne and Lotus corniculatus for gene introgression by Sanjeev V. Raikar Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×106 g-1FW was obtained when cell suspensions were used as the tissue source, with enzyme combination ‘A’ (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×106 g-1FW) of L. corniculatus was achieved from cotyledons also with enzyme combination ‘A’ (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm2 for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.

Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×10⁶ g⁻¹FW was obtained when cell suspensions were used as the tissue source, with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×10⁶ g⁻¹FW) of L. corniculatus was achieved from cotyledons also with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm² for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.