Using RAPD for estimating genetic polymorphism in and phylogenetic relationships among species of the genus Lycopersicon (Tourn.) Mill

RAPD genome analysis of 53 species and cultivars of the genus Lycopersicon (Tourn.) Mill. revealed their high genetic polymorphism (Tourn.) Mill., based on which their phylogenetic relationships were inferred. In total, 248 polymorphic DNA fragments were amplified. Intraspecific polymorphism was maximum (79%) in L. peruvianum and minimum (9%) in L. parviflorum. In general, genome divergence among cross-pollinating tomato species was substantially higher than in self-pollinating species. An UPGMA dendrogram constructed from the RAPD patterns was consisted with the Lycopersicon phylogeny inferred from the molecular data of RFLP, ISSR, and microsatellite analyses and with a classification based on morphological characters. The relationships of taxa within the genus Lycopersicon are discussed.     

RAPD markers for constructing intraspecific tomato genetic maps

The existing molecular genetic maps of the tomato, Lycopersicon spp, are constructed based on isozyme and RFLP polymorphisms between tomato species. These maps are useful for certain applications but have few markers that exhibit sufficient polymorphisms for intraspecific analysis and manipulations within the cultivated tomato. The purpose of this study was to investigate the relative potential of RAPD technology, as compared to isozymes and RFLPs, to generate polymorphic DNA markers within cultivated tomatoes. Sixteen isozymes and 25 RFLP clones that were known to detect polymorphism between L. esculentum and L. pennellii, and 313 random oligonucleotide primers were examined. None of the isozymes and only four of the RFLP clones (i.e., 16%) revealed polymorphism between the cultivated varieties whereas up to 63% of the RAPD primers detected one or more polymorphic DNA fragments between these varieties. All RAPD primers detected polymorphism between L. esculentum and L. pennellii genotypes. These results clearly indicate that RAPD technology can generate sufficient genetic markers exploiting sequence differences within cultivated tomatoes to facilitate construction of intraspecific genetic maps.Abbreviations RFLP restriction fragments length polymorphism - RAPD random amplified polymorphic DNA - PCR polymerase chain reaction - QTLs quantitative trait loci     

Differential response of domestic and wild Lycopersicon species to chilling under low light: growth, carbohydrate content, photosynthesis and the xanthophyll cycle

The response of five Lycopersicon species to 14 days moderate chilling at 10°C under low light (75 μmol m^?2 s^?1) and subsequent recovery was examined by measurements on relative shoot growth rate, leaf dry matter and carbohydrate content, CO₂-exchange and pigment composition. In addition, the susceptibility to dark chilling and temperature dependence of chloroplast electron transport were analyzed by Chl a fluorescence measurements. During 7 days of recovery at 25/20°C subsequent to chilling, the domestic tomato Lycopersiconesculentum (L.) Mill. cv. Abunda exhibited a small capacity for shoot regrowth (39%) compared to the low-altitude wild species L. pimpinellifolium (Jusl.) Mill. PI187002 (82%) and three wild species originating from high altitude: L. peruvianum Mill. LA 385 (92%), L. hirsutum Humb. & Bonpl. LA 1777 (67%) and L. chilense Dunn. LA 1970 (71%). The inter-specific differences in chilling sensitivity at the chloroplast level, analyzed by the decline of the maximum rate of induced Chl fluorescence rise (F_R) after 40 h at 0°C and the temperature at which q_P reached the value 0.5, correlated in general well with the measured differences at whole plant level, measured by the post-chilling regrowth capacity. Chilling resulted in a larger increase in leaf dry matter content in L. esculentum (45%) and L. pimpinellifolium (37%) compared to the high-altitude species (13–16%), which could be attributed to a stronger accumulation of both soluble sugars and starch in mature leaves of the domestic and low-altitude species. Photosynthetic and dark respiration rates during chilling could not account for this difference. The recovery of photosynthesis was better in the high-altitude species. Chl content per unit leaf area decreased more throughout the experiment in the domestic and low-altitude species (63–73%) than in their relatives from high altitude (8–29%). In response to chilling, the domestic and low-altitude species showed an increase in the total xanthophyll cycle pool on Chl basis, whereas the de-epoxidation state of the xanthophyll cycle increased in the high-altitude wild species. Both responses resulted in increased zeaxanthin levels in chilled leaves of all Lycopersicon species.     

Genetic Diversity and Phylogenetic Relationships in the Genus Lycopersicon (Tourn.) Mill. as Revealed by Inter-Simple Sequence Repeat (ISSR) Analysis

Inter-simple sequence repeat (ISSR) analysis was for the first time used to study the genetic diversity and phylogenetic relationships in 54 wild accessions and cultivars of the genus Lycopersicon. Analysis involved 14 ISSR primers homologous to microsatellite repeats and containing additional selective anchor nucleotides. In total, 318 ISSR fragments were amplified for the wild and cultivated tomato genomes. The interspecific polymorphism revealed with the ISSR primers was 95.6%. Species-specific ISSR fragments were detected for each tomato species. The highest number (more than 20) of species-specific fragments were obtained for L. esculentum sensu lato, although the intraspecific variation of ISSR patterns was low. UPGMA cluster analysis was used to construct a dendrogram and to estimate the genetic distances between the species of the genus Lycopersicon; between populations ofL. peruvianum, L. pimpinellifolium, and L. esculentum; and between tomato cultivars. The ISSR-based phylogeny was generally consistent with Lycopersicon taxonomy based on morphological and molecular evidence, suggesting the applicability of ISSR analysis for genotyping and phylogenetic studies in tomato.     

Genetic variation detected with RAPD markers among upland and lowland rice cultivars (Oryza sativa L.)

Genetic variation of nine upland and four lowland rice cultivars (Oryza sativa L.) was investigated at the DNA level using the randomly amplified polymorphic DNA (RAPD) method via the polymerase chain reaction (PCR). Forty-two random primers were used to amplify DNA segments and 260 PCR products were obtained. The results of agarosegel electrophoretic analysis of these PCR products indicated that 208 (80%) were polymorphic. All 42 primers used in this experiment were amplified and typically generated one-to-four major bands. Only two primers showed no polymorphisms. In general, a higher level of polymorphism was found between japonica and indica subspecies while fewer polymorphisms were found between upland and lowland cultivars within the indica subspecies. A dendrogram that shows the genetic distances of 13 rice cultivars was constructed based on their DNA polymorphisms. Classification of rice cultivars based on the results from the RAPD analysis was identical to the previous classification based on isozyme analysis. This study demonstrated that RAPD analysis is a useful tool in determining the genetic relationships among rice cultivars.     

Characterization of genetic diversity of nuclear and mitochondrial genomes in Daucus varieties by RAPD and AFLP

The genetic diversity of nuclear genomes of five Daucus species and seven Daucus carota L. subspecies involving 26 accessions was characterized with random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). AFLP produced more than four times as many discrete bands per reaction compared with RAPD analysis, while both AFLP and RAPD basically led to similar conclusions. The dendrograms constructed with both RAPD and AFLP revealed that all accessions of D. carota were grouped into a major cluster delimited from other Daucus species, in good agreement with the classification by morphological char-acteristics. All accessions of cultivated carrots [(D. carota ssp. sativus (Hoffm.) Arcang.] were clustered in the same group while the variation within D. carota was relatively extensive. Genetic diversity of mitochondrial genomes was also documented with RAPD for the same accessions. The mitochondrial dendrogram differed from that of the nuclear genome, suggesting that nuclear and mitochondrial genomes of some accessions had separate evolutionary histories. Received: 20 September 1997 / Revision received: 19 January 1998 / Accepted: 28 March 1998     

Genetic fingerprinting of pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives using RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used for the identification of pigeonpea [Cajanus cajan (L.) Millsp.] cultivars and their related wild species. The use of single primers of arbitrary nucleotide sequence resulted in the selective amplification of DNA fragments that were unique to individual accessions. The level of polymorphism among the wild species was extremely high, while little polymorphism was detected within Cajanus cajan accessions. All of the cultivars and wild species under study could be easily distinguished with the help of different primers, thereby indicating the immense potential of RAPD in the genetic fingerprinting of pigeonpea. On the basis of our data the genetic relationship between pigeonpea cultivars and its wild species could be established.NCL Communication No. 6062     

Genetic diversity and relationships detected by isozyme and RAPD analysis of crop and wild species of Amaranthus

 Genetic diversity and relationships of 23 cultivated and wild Amaranthus species were examined using both isozyme and RAPD markers. A total of 30 loci encoding 15 enzymes were resolved, and all were polymorphic at the interspecific level. High levels of inter-accessional genetic diversity were found within species, but genetic uniformity was observed within most accessions. In the cultivated grain amaranths (A. caudatus, A. cruentus, and A. hypochondriacus), the mean value of H_T was 0.094, H_S was 0.003, and G_ST was 0.977 at the species level. The corresponding values in their putative wild progenitors (A. hybridus, A. powellii, and A. quitensis) were 0.135, 0.004, and 0.963, respectively. More than 600 RAPD fragments were generated with 27 arbitrary 10-base primers. On average, 39.9% of the RAPD fragments were polymorphic among accessions within each crop species; a similar level of polymorphism (42.8%) was present in the putative progenitors, but much higher levels of polymorphism were found in vegetable (51%) and other wild species (69.5%). The evolutionary relationships between grain amaranths and their putative ancestors were investigated, and both the RAPD and isozyme data sets supported a monophyletic origin of grain amaranths, with A. hybridus as the common ancestor. A complementary approach using information from both isozymes and RAPDs was shown to generate more accurate estimates of genetic diversity, and of relationships within and among crop species and their wild relatives, than either data set alone. Received: 13 March 1997/Accepted: 6 May 1997     

RAPD analysis of the intraspecific and interspecific variation and phylogenetic relationships of <Emphasis Type="Italic">Aegilops</Emphasis> L. species with the U genome

RAPD analysis was used to study the genetic variation and phylogenetic relationships of polyploid Aegilops species with the U genome. In total, 115 DNA samples of eight polyploid species containing the U genome and the diploid species Ae. umbellulata (U) were examined. Substantial interspecific polymorphism was observed for the majority of the polyploid species with the U genome (interspecific differences, 0.01–0,2; proportion of polymorphic loci, 56.6–88.2%). Aegilops triuncialis was identified as the only alloploid species with low interspecific polymorphism (interspecific differences, 0–0.01, P = 50%) in the U-genome group. The U-genome Aegilops species proved to be separated from other species of the genus. The phylogenetic relationships were established for the U-genome species. The greatest separation within the U-genome group was observed for the US-genome species Ae. kotschyi and Ae. variabilis. The tetraploid species Ae. triaristata and Ae. columnaris, which had the UX genome, and the hexaploid species Ae. recta (UXN) were found to be related to each other and separate from the UM-genome species. A similarity was observed between the UM-genome species Ae. ovata and Ae. biuncialis, which had the UM genome, and the ancestral diploid U-genome species Ae. umbellulata. The UC-genome species Ae. triuncialis was rather separate and slightly similar to the UX-genome species.     

Molecular phylogeny in Indian Tinospora species by DNA based molecular markers

The phylogenetic relationships among the three species of Tinospora found in India are poorly understood. Morphology does not fully help to resolve the phylogeny and therefore a fast approach using molecular analysis was explored. Two molecular approaches viz Random Amplified Polymorphic DNA (RAPD) assay and restriction digestion of ITS1-5.8S-ITS2 rDNA (PCR-RFLP) were used to evaluate the genetic similarities between 40 different accessions belonging to three species. Of the 38 random primers used only six generated the polymorphism, while as three out of 11 restriction enzymes used gave polymorphic restriction patterns. The average proportion of polymorphic markers across primers was 95%, however restriction endonucleases showed 92% polymorphism. RAPD alone was found suitable for the species diversions. In contrast PCR- RFLP showed bias in detecting exact species variation. The correlation between the two markers was performed by Jaccard's coefficient of similarity. A significant (r= 0.574) but not very high correlation was obtained. Further to authenticate the results obtained by two markers, sequence analysis of ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) was performed. Three independent clones of each species T. cordifolia, T. malabarica and T. crispa were sequenced. Phylogenetic relationship inferred from ITS sequences is in agreement with RAPD data.     

Proteinase inhibitors I and II in fruit of wild tomato species: Transient components of a mechanism for defense and seed dispersal

The juice of unripe fruit from a wild species of tomato, Lycopersicon peruvianum (L.) Mill., LA 107, contains over 50% of its soluble proteins as the sum of two proteinase inhibitors. These are the highest levels of proteinase inhibitors and highest percentage of soluble proteins as proteinase inhibitors of any plant or animal tissue found to date. Fruit of the modern tomato, L. esculentum Mill., contains only negligible quantities of the two inhibitors. The two proteinase inhibitors in the fruit of L. peruvianum are members of the Inhibitor I and II families previously found in potato tubers and in leaves of wounded potato and tomato plants. The levels of the two inhibitors in the unripe fruit decrease significantly during ripening. Unripe fruit from other wild Lycopersicon species such as L. parviflorum Rick, Kesicki, Fobes et Holle, L. hirsutum Humb. et Bonpe., L. pimpinellifolium Mill., and other lines of L. peruvianum contain moderate levels of the inhibitors that also decrease during ripening. Another wild tomato species, L. pennellii Corr., is similar to L. esculentum in not containing the two proteinase inhibitors in either unripe or ripe fruit. The transient levels of the inhibitors in fruit of wild species indicate that they are present in unripe fruit as defensive chemicals against insects, birds or small mammals and their disappearance during ripening may render them edible to facilitate seed dispersal. High levels of mRNAs coding for Inhibitors I and II in unripe fruit of L. peruvianum, LA 107, indicate that strong promoters may regulate the developmentally expressed proteinase-inhibitor genes in tomato fruit that may have a substantial potential for use in genetic-engineering experiments to enhance the production of large quantities of proteinase inhibitors or other proteins in field tomatoes.Abbreviations poly(A)⁺ mRNA polyadenylated mRNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide electrophoresis Project 1791, College of Agriculture and Home Economics Research Center, Washington, State University     

Characterization of genetic variation in and phylogenetic relationships among diploid<Emphasis Type="Italic"> Aegilops</Emphasis> species by AFLP: incongruity of chloroplast and nuclear data

Intra- and inter-specific genetic variation was investigated in seven diploid Aegilops species using the amplified fragment length polymorphism (AFLP) technique. Of the seven species, the cross-pollinating Aegilops speltoides and Aegilops mutica showed high levels of intraspecific variation whereas the remaining five self-pollinating species showed low levels. Aegilops bicornis, Aegilops searsii and Ae. speltoides formed one cluster in the dendrograms, while Aegilops caudata and Aegilops umbellulata formed another. Relationships among the species inferred were more consistent with the relationships inferred from studies of chromosome pairing in interspecific hybrids, and previous molecular phylogenetic reconstructions based on nuclear DNA, than they were with those based on molecular plasmon analysis, suggesting that the nuclear genome has evolved differently from the cytoplasmic genome in the genus Aegilops.Communicated by J. Dvorak     

Extension of the linkage map in Citrus using random amplified polymorphic DNA (RAPD) markers and RFLP mapping of cold-acclimation-responsive loci

Genetic mapping with RAPD markers has been initiated in Citrus. Reproducible polymorphism of amplified DNA fragments was obtained with approximately half of the 140 random primers tested, revealing 266 segregating loci. These were tested for linkage using 60 BC₁ progeny from an intergeneric cross of Citrus grandis (L.) Osb. x [Citrus grandis (L.) Osb. x Poncirus trifoliata (L.) Raf.]. A core linkage map was constructed that consists of nine linkage groups containing 109 RAPD markers and 51 previously-mapped RFLP and isozyme markers. A further 79 markers that could not be ordered unambiguously because of their allelic constitution were associated with individual linkage groups and are shown in relation to the core map. The core map has a total length of 1192 cM with an average distance of 7.5 cM between loci and is estimated to cover 70–80% of the genome. Loci with distorted segregation patterns clustered on several linkage groups. Individual clusters of loci were skewed in allelic composition toward one or the other parent, usually C. grandis. This relatively-saturated linkage map will eventually be used to identify quantitative trait loci for cold and salt-tolerance in Citrus. As a beginning we have mapped three loci detected by a cold-acclimation-responsive cDNA.     

Phylogenetic analyses within three sections of the genus Vicia

The averaged genomic similarities based on multilocus randomly amplified polymorphic DNA (RAPD) were calculated for eight species representing three sections of the genus Vicia: faba, bithynica and narbonensis. The frequency of appearance of the sequences corresponding to 25 decamers selected at random from genomes of different Fabace species was checked, and a high correlation with the frequency observed for Vicia allowed us to assume their similar weight in typing Vicia species. The RAPD-based similarity coefficients compared with those related to whole genome hybridization with barley rDNA and those based on restriction fragment length polymorphism (RFLP) revealed similar interspecies relationships. The averaged RAPD-based similarity coefficient (Pearson’s) was 0.68 for all the species, and was sectionspecific: 0.43 (bithynica), 0.50 (faba) and 0.73 (narbonensis). The averaged similarity coefficient for V. serratifolia (0.63) placed it apart from the rest (0.75) of its section. The results correspond to the interspecies relationships built upon non-genetic data. The averaged similarity coefficient for particular RAPD was related to the presence and type of tandemly repeated motif in a primer: 0.7–0.8 for heterodimers (GC, AG, CA, GT, CT), 0.5–0.6 for homodimers (CC, GG) and 0.6 for no repeat, indicating the sensitivity of diversity range to the type of target sequences.     

The Genus Syringa: Molecular Markers of Species and Cultivars

RAPD analysis was carried out with 22 accessions of the genus Syringa, including six species, one interspecific hybrid, and 15 cultivars. In total, 512 polymorphic fragments were detected; species-specific and cultivar-specific markers were identified. For the first time, genetic polymorphism and genome similarity coefficients were estimated and phylogenetic relationships were established for the genus Syringa.     

Random amplified DNA polymorphism of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. cultivars

The polymorphism, similarities and relationships among Nicotiana tabacum L. cultivars were assessed with RAPD analyses. One hundred and forty-nine bands were detected, of which 94 were polymorphic (63.1 %). A primer distinguishing all of the tested cultivars was found. High similarity between cultivars was revealed, and cultivar relationships were estimated through cluster analysis (UPGMA) based on RAPD data.The experiments in this study were carried out at the South Center Tobacco Breeding Research of China; the expense was provided by Yunnan Tobacco Company.     

Molecular characterization of endophytic strains of Fusarium verticillioides (= Fusarium moniliforme) from maize (Zea mays. L)

The species Fusarium verticillioides (= F. moniliforme) is often found in maize seeds, constituting an important source of inoculum in the field. Fusarium spp., associated with symptomatic and asymptomatic plants, may be a primary causal agent of disease, a secondary invader or an endophyte. In the present work, endophytic fungi were isolated from two populations of Zea mays (BR-105 and BR-106) and their respective inbred lines. Within different inbred lines of maize, Fusarium was found at a frequency of 0 to 100% relative to the number of total isolated fungi. The frequency with which the genus occurred was practically the same in the two field sites (around 60%). Twenty-one F. verticillioides strains were analysed using the random amplified polymorphic DNA (RAPD) technique, employing 10 random primers. Variability analysis of endophytic isolates via RAPD showed genome polymorphism taxa of species around 60%. Endophytic isolates were clustered by their sites of origin. RAPD analysis clustered the endophytic isolates by their maize inbred lines hosts (Mil-01 to Mil-06), whereas at site A they clustered into two major groups related to the maize gene pool (BR-105 or BR-106 population). All strains isolated from seeds collected in Site A, except strains L9 and L10, were sub-grouped according to maize inbred lines. The analysis showed a discrete sub-grouping at site B. Results obtained here could be explained by a co-evolution process involving endophytic isolates of F. verticillioides and maize inbred lines.     

Polymorphism and Divergence of Multilocus DNA Markers in Sibling Species Chironomus riparius Meigen and Chironomus piger Strenzke (Diptera,Chironomidae)

Intra- and interspecific variation and divergence of multilocus markers for genomic DNA of the sibling species from the thimmi group,Chironomus riparius and C. piger, were studied by PCR with arbitrary primers (RAPD). A high level of RAPD polymorphism was determined in both laboratory and natural populations of these species. The genetic distances were estimated between the C. riparius populations and between the sibling species C. riparius and C. piger. The genetic distance between C. riparius andC. piger was 4 to 5 times higher than that between the C. riparius populations. A comparison of the variation and divergence for the RAPD markers with those for other genomic markers—enzyme-coding genes and chromosomes (gene linkage groups)—showed that different components of the genome differed in their contribution to the genome divergence.     

Resistenz verschiedener Kultur- und Wildtomatenpflanzen (Lycopersicon spp.) gegenüber Botrytis cinerea Pers.

Resistance of different cultivated and wild tomato plants (Lycopersicon spp.) to Botrytis cinerea Pers. 20 provenances of different cultivated and wild tomato plants (Lycopersicon spp.) were screened for resistance to Botrytis cinerea Pers. using an in vitro-leaf necrosis test. The Botrytis resistance decreased with increasing age of the leaves corresponding to their insertion height (relative youth resistance respectively senescence susceptibility). The 6 B. cinerea-isolates used for inoculation differed significantly in virulence. With increasing inoculum age a virulence reduction of the various B. cinerea-isolates occurred. Within the investigated test plant collection 2 wild species –L. columbianum and L. hirsutum– proved to be resistant in each stage of development to all B. cinerea-isolates and additionally showed field resistance.     

LTR-retrotransposons Tnt1 and T135 markers reveal genetic diversity and evolutionary relationships of domesticated peppers

Plant genetic resources often constitute the foundation of successful breeding programs. Pepper (Capsicum annuum L.) is one of the most economically important and diversely utilized Solanaceous crop species worldwide, but less studied compared to tomato and potato. We developed and used molecular markers based on two copia-type retrotransposons, Tnt1 and T135, in a set of Capsicum species and wild relatives from diverse geographical origins. Results showed that Tnt1 and T135 insertion polymorphisms are very useful for studying genetic diversity and relationships within and among pepper species. Clusters of accessions correspond to cultivar types based on fruit shape, pungency, geographic origin and pedigree. Genetic diversity values, normally reflective of past transposition activity and population dynamics, showed positive correlation with the average number of insertions per accession. Similar evolutionary relationships are observed to that inferred by previous karyosystematics studies. These observations support the possibility that retrotransposons have contributed to genome inflation during Capsicum evolution.