Amplified DNA Fingerprinting Research Articles

Population genetic structure of the pioneer weed Cyperus ligularis in urban areas

AbstractHumans change the environment according to their need, causing modifications and affecting the local biodiversity. This work applied a set of dominant markers to test if urban populations of Cyperus ligularis L. (Cyperaceae) present less genetic diversity compared to forest/non‐urban populations, and if these environmental differences (land use) influence gene flow and population structure. Six available populations of C. ligularis were collected in different types of environments in Recife (Brazil). Overall 15 primers of DNA Amplification Fingerprinting and Inter Simple Sequence Repeat were applied and analyzed by proper statistics. The results revealed moderate genetic diversity, significant pairwise gene flow (Nem), and low differentiation between populations (FST). Land use had no direct correlation with genetic diversity or structure, however, an insular population showed higher differentiation from the remaining. Urban environments may not influence gene flow among C. ligularis populations in such environments; therefore, the genetic diversity is homogeneous in the analyzed area.

Random Amplified Polymorphic DNA (RAPD) and Derived Techniques.

Understanding biology and genetics at molecular level has become very important for dissection and manipulation of genome architecture for addressing evolutionary and taxonomic questions. Knowledge of genetic variation and genetic relationship among genotypes is an important consideration for classification, utilization of germplasm resources, and breeding. Molecular markers have contributed significantly in this respect and have been widely used in plant science in a number of ways, including genetic fingerprinting, diagnostics, identification of duplicates and selection of core collections, determination of genetic distances, genome analysis, development of molecular maps, and identification of markers associated with desirable breeding traits. The application of molecular markers largely depends on the type of markers employed, distribution of markers in the genome, type of loci they amplify, level of polymorphism, and reproducibility of products. Among many DNA markers available, random amplified polymorphic DNA (RAPD) is the simplest, is cost-effective, and can be performed in a moderate laboratory for most of its applications. In addition, RAPDs can touch much of the genome and has the advantage that no prior knowledge of the genome under research is necessary. The recent improvements in the RAPD technique like arbitrarily primed polymerase chain reaction (AP-PCR), sequence characterized amplified region (SCAR), DNA amplification fingerprinting (DAF), sequence-related amplified polymorphism (SRAP), cleaved amplified polymorphic sequences (CAPS), random amplified microsatellite polymorphism (RAMPO), and random amplified hybridization microsatellites (RAHM) can complement the shortcomings of RAPDs and have enhanced the utility of this simple technique for specific applications. Simple protocols for these techniques are presented along with the applications of RAPD in genetic diversity analysis, mapping, varietal identification, genetic fidelity testing, etc.

Establishment and characterization of a new embryonic cell line from Helicoverpa armigera (Hübner).

Here, a new cell line, Ha168, was established from Helicoverpa armigera eggs and has been stably subcultured for over 30 passages in TNM-FH medium supplemented with 10% fetal bovine serum. The cell line consists of round and spindle-shaped cells and several giant cells. The round cells, with a cell diameter of 14.30 ± 2.804μm, account for 77% of the cells. DNA amplification fingerprinting, random amplified polymorphic DNA analysis, and analysis of the mitochondrial cytochrome c oxidase subunit I gene confirmed that the Ha168 cells were derived from H. armigera. Karyotype analysis revealed the average chromosome number of Ha168 cells to be 71. Growth curves at passage 25 were determined and demonstrated that the cell population doubling time is 56.8h. No mycoplasma contamination was detected in the cell line. Ha168 cells can be infected by recombinant baculovirus AcMNPV-EGFP, and exogenous protein expression level in this cell line is 70% of that in the Sf9 cell line.

Advances in monitoring soil microbial community dynamic and function.

Microorganisms are vital to the overall ecosystem functioning, stability, and sustainability. Soil fertility and health depend on chemical composition and also on the qualitative and quantitative nature of microorganisms inhabiting it. Historically, denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE), single-strand conformation polymorphism, DNA amplification fingerprinting, amplified ribosomal DNA restriction analysis, terminal restriction fragment length polymorphism, length heterogeneity PCR, and ribosomal intergenic spacer analysis were used to assess soil microbial community structure (SMCS), abundance, and diversity. However, these methods had significant shortcomings and limitations for application in land reclamation monitoring. SMCS has been primarily determined by phospholipid fatty acid (PLFA) analysis. This method provides a direct measure of viable biomass in addition to a biochemical profile of the microbial community. PLFA has limitations such as overlap in the composition of microorganisms and the specificity of PLFAs signature. In recent years, high-throughput next-generation sequencing has dramatically increased the resolution and detectable spectrum of diverse microbial phylotypes from environmental samples and it plays a significant role in microbial ecology studies. Next-generation sequencings using 454, Illumina, SOLiD, and Ion Torrent platforms are rapid and flexible. The two methods, PLFA and next-generation sequencing, are useful in detecting changes in microbial community diversity and structure in different ecosystems. Single-molecule real-time (SMRT) and nanopore sequencing technologies represent third-generation sequencing (TGS) platforms that have been developed to address the shortcomings of second-generation sequencing (SGS). Enzymatic and soil respiration analyses are performed to further determine soil quality and microbial activities. Other valuable methods that are being recently applied to microbial function and structures include NanoSIM, GeoChip, and DNA stable staple isotope probing (DNA-SIP) technologies. They are powerful metagenomics tool for analyzing microbial communities, including their structure, metabolic potential, diversity, and their impact on ecosystem functions. This review is a critical analysis of current methods used in monitoring soil microbial community dynamic and functions.

Establishment of two midgut cell lines from the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).

Two cell lines were generated from larval midguts of Spodoptera frugiperda and have been 26 passaged over 50 times. The CT/BCIRL-SfMG1-0611-KZ line was established from 27 trypsinized, minced whole midgut tissues: the CT/BCIRL-SfMG-0617-KZ line from isolated 28 midgut muscle tissue (containing some residual epithelial cells). Additional midgut cultures were 29 generated from isolated epithelial cells; some passaged not more than three times, which grew 30 very slowly and survived longer than 1 year. The continuously replicating cell lines contain 31 firmly adhering cells with different morphologies, including elongated, spherical, and/or 32 rectangular. The mean diameters of these cell lines are 9.3 ± 4.0 μm (SfMG1-0611) and 9.2 ± 3.9 33 μm (SfMG-0617). Growth curves for the two lines have relatively lengthy doubling times of 73.9 34 h and 50.4 h for SfMG1-0611 and SfMG-0617, respectively. We confirmed the identity of these 35 lines using DNA amplification fingerprinting (DAF-PCR) and noted that the DNA patterns for 36 each cell line were similar to their host tissues but distinctly different from other cell lines or 37 tissues from different insect species. Amplification of genomic DNA with species-specific 38 primers yielded DNA fragments of the expected sizes and with sequences nearly identical to 39 those from the S. frugiperda genome. Both cell lines were exposed to selected Bt Cry proteins 40 with minimal impact. These lines are currently available to researchers worldwide.

Next-generation cell lines established from the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).

The fall armyworm, Spodoptera frugiperda (Sf), is a polyphagous lepidopteran herbivore that consumes more than 80 plant species, including many economically important crops, such as corn, soybeans, and sorghum. While already a serious pest in the Americas, it was recently introduced into Africa, India, and China. Because of its high economic costs in the New World and the continent-wide damage potentials in Africa, research to develop advanced pest management technologies is necessary. We are supporting this need by developing novel, next-generation insect cell lines from targeted tissues. Cell lines, such as these, will boost insecticide discovery programs and lead to innovative pest management solutions. Here, we report on the establishment of 16 new cell lines from larval S. frugiperda tissues: nine from the central nervous system, three from the aorta, and four from the testes. We confirmed the identities of the cell lines by DNA amplification fingerprinting polymerase chain reaction, determined their doubling times from growth curves, and described cell types via microscopy. We also developed 16 sublines from three neuronal cell lines.

Molecular Tools for Understanding Landscape Genetics and the Population Genetic Effects of Habitat Restoration on Butterflies

Habitat corridor construction is an important technique for re-establishing connections between fragmented habitats. Yet, the effectiveness of habitat corridors to increase gene flow among fragmented populations is not well studied. In 2008 and 2009, a prairie habitat corridor was created in the Green River watershed of south central Kentucky, USA to protect water quality and encourage movement of native wildlife as part of the US Environmental Protection Agency Conservation Reserve Enhancement Program (CREP). In 2008, prior to the establishment of the habitat corridor, we documented the population genetic structure of 6 butterfly species (Chlosyne nycteis, Cupido comyntas, Phoebis sennae, Phyciodes tharos, Pterourus glaucus, and Pterourus troilus) with different habitat requirements and different expected responses to corridor construction using Randomly Amplified DNA Fingerprint (RAF) markers. STRUCTURE analysis of these markers subdivided each butterfly species into 2 to 8 subpopulations in the Green River watershed. By collecting data in the early stages of habitat restoration, we have established a baseline to compare with data obtained after the corridor has matured to determine the population genetic effects on previously isolated butterfly populations and to provide information about the conservation value of habitat corridors in general.

The Taxonomy and Population Structure of the Buckeye Butterflies (GenusJunonia, Nymphalidae: Nymphalini) of Florida, USA

The buckeye butterflies (genus Junonia) that occur in Florida, USA have been an ongoing taxonomic challenge for over 100 years. Schwartz (1987. Milwaukee Pub. Mus. Contrib. Biol. Geol. 73: 21) described the situation succinctly: “No other lepidopteran on the Florida Keys nor in south Florida, presents the taxonomic problems as Junonia.” The current consensus recognizes 3 different forms of Junonia in Florida based on morphological characters, but there is a general lack of consensus regarding the appropriate scientific names for each form. Similarities between the species that occur in Florida, intraspecific variation, and possible hybridization between species have often made it challenging to identify specimens, define the population structure of Junonia butterflies, or to understand the relationships of these butterfly populations to those elsewhere in the New World. We use a combination of morphological characters, mitochondrial cytochrome oxidase I barcodes, nuclear wingless DNA sequences, and randomly amplified DNA fingerprints from Junonia from Florida, the Caribbean, and North and South America to resolve issues of taxonomy and population structure in this genus. We conclude that the common buckeye (J. coenia), the mangrove buckeye (J. neildi), and the tropical buckeye (J. zonalis) occur in Florida and that hybridization between these species takes place in this region.

MOLECULAR DIVERSITY AND MORPHOMETRIC ANALYSIS OF CORBICULA FLUMINEA (MÜLLER, 1774) (BIVALVIA: CORBICULIDAE) FROM THE RIVER NILE, EGYPT

Corbicula fluminea is a spread freshwater bivalve with high nutritional and medical values. The present study aimed to investigate the molecular diversity within the collected four morphs of C. fluminea from the River Nile in Egypt by using RAPD-PCR biomarkers. The genetic polymorphism among their amplified DNA fingerprints of the four forms of C. fluminea population was performed by applying an available online program; Jaccard-Needham dissimilarity distance. These four morphotypeshells were also statistically distinguished morphologically and morphometrically depending on their colour pattern, and measurements of their lengths, heights and widths. Furthermore, the cluster analysis of the phenogram software program was documented for determining the phylogenetics. The biometric parameters and genetic distances manifested two major clusters of C. fluminea. The first major cluster had only one variety {Morph-I} with the highest ratio of genetic distance 0.9 reflecting a great genetic distance with other three varieties. The second major cluster has three polymorphics and again divided into two minor clusters. One minor cluster, further divided into two varieties {Morph-III & Morph-IV} and other having only one variety {Morph-II}. Hence, the study assessed and established a relatively DNA barcode reference of that molecular clam diversity.

A cell clone strain from Mythimna separata (Lepidoptera: Noctuidae) highly susceptible to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata NPV (MsNPV).

In this study, we describe a cell line, Ms-10C, cloned from the line QAU-Ms-E-10 (simplified Ms-10), an embryonic line from Mythimna separata. The cloned cell line was significantly more sensitive to nucleopolyhedrovirus (NPV). Ms-10C cells were mainly spherical with a diameter of 14.42±2.23μm. DNA amplification fingerprinting (DAF) confirmed the profile of PCR-amplified bands of the cloned cell line was consistent with those of the parental cell line, Ms-10. The sequencing result of the mitochondrial cytochrome c oxidase I (mtCO I) fragment confirmed that the amplified 636-bps mtCOI fragment was 100% identical to that of M. separata. Its chromosomes exhibited the typical characters of lepidopteran cell lines. Its population doubling time was 42.2h at 27°C. Ms-10C was more sensitive than Ms-10 to both Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata nucleopolyhedrovirus (MsNPV). At 4d post infection, the infection rates of two viruses reached 94.2 and 92.3%, respectively. The availability of this cell clone strain will provide a useful tool for the basic research on nucleopolyhedrovirus and for potential application in expression of recombinant proteins with baculovirus expression vector system.

Морфолого-генетический анализ ценопопуляций Stachys sylvatica (Lamiaceae), произрастающих в горах Южной Сибири

Stachys sylvatica – реликт третичных широколиственных лесов гор Южной Сибири. Проведен анализ модификационной и генетической изменчивости признаков 11 ценопопуляций Stachys sylvatica, произрастающих в сообществах гор Южной Сибири. Выявлено, что данный вид встречается в хвойных (пихтовых, сосновых, кедровых, кедрово-пихтовых и кедрово-сосновых), осиновых, липовых лесах, а также в прирусловых ивняках и черемушниках. Климатический ареал вида ограничен годовым количеством осадков 500–1250 мм, суммами активных температур 1600–2050 °С и высотным распространением от 150 до 700 м над ур. м. При изучении состояния ценопопуляций S. sylvatica определяли численность, проективное покрытие, оценивали морфологическую изменчивость вегетативных и генеративных признаков. Для установления достоверности различий между среднепопуляционными значениями одноименных признаков использовали однофакторный дисперсионный анализ. Выявлено, что большинство морфометрических признаков характеризуются средним и высоким уровнем изменчивости. Генетическую вариабельность вида выявляли с помощью метода RAF-PCR (Randomly Amplified DNA Fingerprinting). Проведенный анализ генетической изменчивости ценопопуляций Stachys sylvatica, показал, что для изучаемого вида характерен очень высокий уровень внутрипопуляционного полиморфизма. Максимальный уровень генетической изменчивости отмечен в ценопопуляции, произрастающей на границе ареала. Генофонды всех изученных ценопопуляций характеризуются удовлетворительным состоянием и могут самостоятельно воспроизводиться. Все изученные ценопопуляции чистеца лесного слабо дифференцированы и не проявляют тенденции к образованию новых таксонов. Причем, сходную генетическую структуру обнаруживают ценопопуляции, произрастающие не только в разных частях ареала, но и в сообществах, кардинально различающихся по экологическим и фитоценотическим факторам. При усилении антропогенного воздействия на ценопопуляции наблюдается увеличение размеров генеративных органов Stachys sylvatica. На основе анализа морфологических признаков S. sylvatica выявлено разделение совокупности особей по географическому принципу. Выявлено, что данный вид обладает сравнительно широкой экологической амплитудой и слабой конкурентной способностью. Может поддерживать высокий уровень полиморфизма в стрессовых для других неморальных реликтов условиях. Характеризуется высоким уровнем внутрипопуляционной генетической изменчивости и слабой степенью дифференциации популяций.

Establishment and characterization of a new cell line of Chilo suppressalis Walker (Lepidoptera: Pyralididae).

A new cell line, designated as ZJBIQ-Chsu-I, was initiated from the fat body of larval Chilo suppressalis (Walker) (Lepidoptera: Pyralididae) in TNM-FH insect medium containing 15% fetal bovine serum. The polygonal cells (65.6%) were predominant among various cell types, and the diameter range was from 12.63 to 22.50 μm. The cell line showed a typical lepidopteran chromosome pattern ranging from 108 to 136 chromosomes in the majority of the cells. The population doubling time (PDT) of the cell line at the 15th passage was 62 h. This cell line was found to be susceptible to Spodoptera exigua nuclear polyhedrosis virus (SeNPV). By the DNA amplification fingerprinting polymerase chain reaction (DAF-PCR) technique, it was confirmed that cell line ZJBIQ-Chsu-I really originated from C. suppressalis.

Genetic and epigenetic integrity assessment of acclimatised papaya plants regenerated directly from shoot-tips following short- and long-term cryopreservation

A vitrification-based cryopreservation protocol was applied to in vitro sourced shoot-tips of four genotypes of Carica papaya; two female (70 and Z6) and two male (B2 and B4). Regeneration of ~58 % (70) and ~59 % (Z6) was recorded for the female genotypes confirming previously published results. Regeneration was at ~77 and ~53 % for the two male genotypes B2 and B4 respectively. Cryo-tube storage and regeneration was tested after 2–18 months storage in one male (B2) and one female (70) genotype. Regeneration post cryo-storage was similar to 1 h exposure to liquid nitrogen. Individual shoot-tips from the two female and two male genotypes were grown into complete in vitro plants, potted and acclimatised without micropropagation to provide material for randomly amplified DNA fingerprinting (RAF) and amplified DNA methylation polymorphism (AMP) analysis of multiple individuals from in vitro control, plant vitrification solution 2 (PVS2) cryoprotectant control and short (1 h) and long-term cryopreservation treatment plants. No variations were detected for genotype Z6 control and treatment individuals and no RAF variations were detected in any individuals of genotype B2. Small numbers of RAF and AMP variations were detected in some individuals from genotypes B2 (AMP variation only), B4 and 70, but these were also found in controls. Genotype 70 showed the greatest level of variation; genomic DNA variation (RAF) was detected in control and cryopreservation treatment individuals, and the PVS2 control group was the only treatment group without variations for the respective AMP analysis. The variations observed could not be correlated with any phenotypic characteristics 2 months after acclimatisation.

A new insect cell line from pupal ovary of Spodoptera exigua established by stimulation with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)

A continuous cell line derived from the pupal ovary of Spodoptera exigua was established by treating primary cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Three days after treating cells with 3.0 μg/ml of MNNG, the cells formed a monolayer and were initially subcultured 60 d after the MNNG was removed, followed by subculturing for 30 passages. The established cell line, designated IOZCAS-Spex 12, consisted of a mixture of three types of cells, including spherical, spindle-shaped, and oval cells. The population doubling time of the cell line during its logarithmic growth phase was found to be 71 h. DNA amplification fingerprinting polymerase chain reaction analysis confirmed that the new cell line originated from S. exigua. Susceptibility of IOZCAS-Spex 12 cells to infection by certain nucleopolyhedroviruses was investigated. The results showed that the cell line was highly susceptible to infection by S. exigua nucleopolyhedrovirus and Autographa californica multiple nucleopolyhedrovirus, slightly susceptible to infection by Spodoptera litura nucleopolyhedrovirus, and not susceptible to infection by Helicoverpa armigera nucleopolyhedroviruses or Hyphantria cunea nucleopolyhedroviruses. The results of this study suggest that MNNG treatment may overcome existing limitations to obtaining continually proliferating cells and may open up the possibilities for immortalizing isolated insect cells.

STORAGE STABILITY USING CRYOPRESERVATION: A CASE STUDY IN PAPAYA

ISHS XXVIII International Horticultural Congress on Science and Horticulture for People (IHC2010): III International Symposium on Plant Genetic Resources STORAGE STABILITY USING CRYOPRESERVATION: A CASE STUDY IN PAPAYA

DETECTION OF MOLECULAR MARKERS ASSOCIATED WITH SALT TOLERANCE IN ALFALFA (Medicago sativa L.)

Alfalfa (Medicago sativa L.) is one of the world's most valuable forage legumes. It is grown for hay, pasture and silage, and is valued highly as a livestock feed. It is characterized by adaptability to wide range of soils and growing conditions, flexibility in being able to grow alone or mixed with grasses and high yields. It is the most cultivated forage legume due to its ability to fix atmospheric nitrogen and its high protein content. Soil salinization significantly limits crop production and consequently has negative impact on food security. It is one of the major abiotic stresses that affects crop productivity and quality and has been described as one of the most serious threats to agriculture and the natural status of the environment. Increased salinization of arable land is expected to have devastating global effects, resulting in a 30% land loss within the next 25 years and up to 50% by the year 2050 (Lorenzo et al., 2007). The analysis of the genetic variability within and among populations of cultivated alfalfa can assess future risk of genetic erosion and help in the development of sustainable conservation and genetic improvement strategies. Successful assessment of the genetic diversity of alfalfa has been hampered by the statistical methods available (Stanford, 1951; Flajoulot et al., 2005). Alfalfa species are composed of ecotypes, population complexes adapted to the environment of a given climatic region or to definite habitats within a region (Helmy et al., 2003). Environmental constraints represent the most limiting factors for agricultural productivity and play a major role in the distribution of plant species across different types of environments. Environmental factors, such as drought and salinity are responsible for significant yield reductions. Developing cultivars that tolerate abiotic stresses is one of the major goals of breeding programs of alfalfa. Molecular markers can assist these programs by identifying the important traits, helping in screening the genotypes and selecting them. Molecular markers can be identified by a range of molecular techniques such as restriction fragment length polymorphisms (RFLPs), randomly amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphism (AFLP), DNA amplification fingerprinting (DAF), sequence characterized amplified regions (SCARs), microsatellites (SSR) etc, (Lin et al., 1996). Also, there are several different DNA analytical procedures that have been used to identify, characterize and determine genetic diversity among cultivars. AFLP is one of the most the recently DNA analysis procedures, which combines assay flexibility with a high degree of sensitivity and reproducibility to yield significantly more information about the plant genome under study than other techniques (Lin et al., 1996). AFLP is a method for genotyping individuals for a large number of loci using a minimal number of PCR reactions. AFLP markers are efficient tools for estimating genetic similarity in plant species and effective management of genetic resources. They are a reliable method of genetic fingerprinting and have been successfully used for characterization and evaluation of genetic relationships in several species (Vos et al., 1995; Neqi et al., 2000). The objectives of this study were to test the most tolerant and the most sensitive alfalfa (Medicago sativa L.) genotypes, their F1, and F2 under salt condition for some yield-related traits and obtain molecular genetic markers associated with salt tolerance by bulked segregant’s analysis technique using RAPD, ISSR and AFLP analysis.

5. Molecular profiling of bacterial DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue: a comparative study

There is increasing use of FFPE tissues for molecular profiling. We sought to determine the effects of fixation on our ability to detect bacterial DNA in tissues. Aim To investigate the effect of FFPE, and two de-paraffinisation methods on DNA yields, bacterial DNA amplification and molecular fingerprinting. Methods Intestinal tissue and corresponding gut microflora were collected from mice and chickens. Triplicate samples from 14 animals were either snap frozen at -80°C (FS); or fixed in 10% formalin prior to processing. The FFPE samples were de-paraffinised using either the traditional method (TM) or a microwave method (MW). DNA was extracted from all tissues using a modified commercial kit protocol. DNA yields were determined, and amplification of bacterial 16S rDNA fragments compared. Denaturing gradient gel electrophoresis (DGGE) was used to generate ‘fingerprints’ of PCR amplicons to obtain an estimate of number of species of gut microbiota present. Results DNA yields from FFPE-TM and -MW tissues were 11% and 35% respectively of those from FS. 16S PCR products were obtained from 93% of FS, 7% of FFPE-TM and 57% of FFPE-MW samples. DGGE profiles showed FS yielded PCR fingerprints with the most bands (avg 22 bands) followed by FFPE-MW (avg 3 bands). Conclusion Compared to FS, FFPE tissue yields less bacterial DNA, and is less likely to enable the generation of PCR amplicons or DGGE bands for molecular profiling. The MW method yielded twice the DNA of TM. The amplification success of the 260 bp 16S rDNA fragment is poor when DNA has been extracted from FFPE, regardless of de-paraffinisation method.

Genetic Diversity Among Wheat Cultivars Using Molecular Markers

The objective of this study was to compare amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), and DNA amplification fingerprinting (DAF) marker systems for estimating genetic diversity among 13 Iranian wheat (Triticum aestivum L.) cultivars through average expected heterozygosity (Hav), sum of effective number of alleles (SENA), and marker index (MI). The AFLP markers had the highest values for these parameters. Correlations of RAPD marker data with those obtained using AFLP markers were higher than those obtained using DAF. Mantel correspondence test did not show significant correlations among dendrograms obtained from the three assays. Because of the large size of the wheat genome, different assays detect polymorphism in different genomic regions. The averages of similarities based on Jaccard's coefficient were high for the three assays. We concluded that AFLP markers showed the highest efficiency in estimating genetic diversity in bread wheat germplasm. High level of genetic similarities among Iranian agronomically important bread-wheat cultivars make it necessary to broaden the genetic base of Iranian wheat germplasm.

PAPAYA AS A MODEL TROPICAL FRUIT SPECIES FOR THE DEVELOPMENT OF CONSERVATION AND BREEDING TECHNOLOGIES

Papaya (Carica papaya) has been used in our laboratory with a range of technologies for ex situ conservation of germplasm. It represents a good model species, as it has been the subject of major research efforts in tissue culture and biotechnology. We have investigated seed storage behaviour via desiccation curves based on accurate water activity measurements and a range of storage environments from liquid nitrogen to 15°C/15% RH. Treatments have been optimised to break dormancy and to ensure germination after seed storage. Twenty years of accumulated experience in papaya cell and tissue culture has facilitated our investigation of cryopreservation protocols, including vitrification based techniques, encapsulation and slow freezing, on a range of in vitro tissues from shoot tips to somatic embryos. This research has led to the development of optimum protocols for ex situ conservation of papaya germplasm, both as seed and vegetative tissue which have application to other tropical fruit species. Plants that were recovered post-cryopreservation have been screened for changes in genomic DNA and DNA methylation using the Polymerase Chain Reaction (PCR) based Randomly Amplified DNA Fingerprinting (RAF) and Amplified DNA Methylation Polymorphism (AMP) techniques, and are being evaluated in field plantings. Similarly, work on embryo culture techniques in our lab have contributed to the success of intergeneric breeding programs. Efficient molecular markers have been employed to assess genetic diversity within and between papaya genotypes and related Vasconcella species.

Establishment of a cell line from Chrysodeixis chalcites permissive for Chrysodeixis chalcites and Trichoplusia ni nucleopolyhedrovirus

A new cell line was established from the embryos of the insect Chrysodeixis chalcites (Lepidoptera, Noctuidae, Plusiinae). The cell line contains several morphologically different cell types and was distinguished from three other lepidopteran cell lines propagated in the laboratory by DNA amplification fingerprinting. The cultured cells, which we officially named WU-CcE-1 cells, were permissive for infection by C. chalcites nucleopolyhedrovirus (ChchNPV) and large numbers of occlusion bodies were produced that retained their infectivity for C. chalcites larvae. The CcE-1 cells were also permissive for Trichoplusia ni single nucleopolyhedrovirus (TnSNPV). ChchNPV could be passaged in these cells for at least four passages indicating that budded virus production was supported. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Helicoverpa armigera (Hear) NPV both induced apoptosis in these cells. The results obtained indicate that the CcE-1 cell line will be a useful tool in the study of both ChchNPV and TnSNPV.