Genetic divergence among gerbera accessions evaluated by RAPD

Genetic diversity was evaluated by RAPD markers and morpho-agronomic characters for a total of 42 accessions of Barberton daisy (Gerbera jamesonii) consisting of 29 commercial and 13 wild accessions. A total of 74 polymorphic bands were obtained employing a set of 12 primer pairs. The average genetic similarity coefficient for the 42 accessions, evaluated by Jaccard index was 0.55 ranging from 0.28 to 1.00. The genetic structure found among Barberton daisy accessions was evaluated by hierarchic classification analyses and UPGMA modeling, revealing six clusters of genotypes where two of them include the wild accessions and the remaining four including commercial material, except for wild genotype number 9. Shannon (H′) index was calculated using the molecular markers to investigate the genetic variation among the Gerbera accessions and showed higher values for the commercial cluster in comparison to the values obtained for the individuals from the non-commercial cluster, namely 0.34 versus 0.27, respectively. Therefore, both calculated indices (Jaccard and Shannon) indicated the presence of higher genetic variation among commercial accessions in comparison to the cluster representing non-commercial accessions, suggesting that genetic breeding programs may focus on commercial accessions to recombine interesting genotypes with commercially important and marketing-desired characteristics.     

Assessment of the stability and adaptability of waxbloom and waxless pea (Pisum sativum L.) mutant lines

Genotype–environment interactions, stability and adaptability for plant height, number of pods/plant, number of seeds/plant and seed weight/plant were analyzed by means of the model proposed by Eberhart and Russell (1966) in two cultivars and 22 mutant lines of field peas (Pisum sativum L.). The experimental design was the randomized block with three replicates. The genotypes were evaluated in 3 years. The stability parameters were: regression coefficient, variance due to regression and coefficient of determination. Differences in the response to environment were found among genotypes for the characters studied. The prevalent part of the investigated mutants and both cultivars reveal specific adaptation responding differently to environments, thus representing an initial plant material for developing individual targeted breeding programs.     

Comparison of the use of morphological,protein and DNA markers in the genetic characterization of Iranian wild Prunus species

In this study, the genetic diversity of four Iranian wild Prunus species including Prunus eleagnifolia, Prunus hauskenchtii, Prunus scoparia and Prunus lycioides were investigated using morphological, protein and DNA markers. DNA markers included nuclear and chloroplast SSRs and self-incompatibility (S) allele amplification. At the morphological level, leaf width showed significant differences between the four wild Prunus species. Concerning fruit and kernel characters, their values are relatively similar indicating the high degree of homoplasy described in Prunus. Nuclear SSR markers have been the most abundant markers with a higher polymorphism in comparison with morphological, protein and chloroplast SSR markers. Results also indicated the high variability present in the S locus. On the other hand, the correlation between the clustering based on DNA markers and protein were in general low. Dendogram performed using nuclear and chloroplast SSR indicated a more diffuse clustering between the wild almond species probably due to the natural introgression of genes observed in these wild almond species. Data from the analysis of the total protein seems to be more accurate to establish taxonomy relationships in these very close wild species.     

Cryopreservation of coriander (Coriandrum sativum L.) somatic embryos using sucrose preculture and air desiccation

An efficient protocol for cryopreservation of somatic embryos of Coriandrum sativum, an important spice and medicinal herb, was developed. The successful cryopreservation procedure utilized embryo clumps (ECs) comprised of 3–4 somatic embryos at the globular or heart-shape stage. These ECs were precultured for 3 days on medium supplemented with 100 g/L sucrose, desiccated under the current of sterile air for 100 min, then sealed in cryovials and plunged directly into liquid nitrogen. Preliminary incubation on sucrose-enriched medium (100 g/L) improved both desiccation- and cryo-tolerance of ECs compared to medium with normal sucrose content (30 g/L) and enhanced embryo formation after cryopreservation. The regrowth after cryopreservation and average number of new embryos developed from cryopreserved ECs were retained at the level of the untreated control (98% and 13 embryos per clump, respectively). Both normal and abnormal plants were produced from control and cryopreserved cultures, indicating that appearance of abnormalities was not related to cryopreservation. The regenerants with normal phenotype showed the same peaks of relative DNA content regardless of cryopreservation. The results suggest that simple desiccation method is effective for cryopreservation of coriander somatic embryos with subsequent regeneration.     

Study of genetic diversity in Chamomile (Matricaria chamomilla) based on morphological traits and molecular markers

Chamomile is one of the most important medicinal plants in the world trade that has many applications in drug and sanitary industrials. In order to evaluate the genetic diversity of different chamomile landraces based on morphological and molecular markers, 20 landraces were collected from different area of Iran. In addition to that, five populations imported from European were examined. The augmented design with four blocks and five controls were used to assess morphological traits. The RAPD method was utilized for evaluating the genetic diversity. Results showed that the economical yield, the number of flowers in plant, and the essential oil content had maximum variance coefficient. The flower's diameter and height had minimum variance coefficient. According to the cluster analysis on both morphological and molecular markers, 25 populations were classified into 5 clusters, but the population intra-groups were different. From 29 reliable primers that were used, 369 bands were detected and from which 314 (85.44%) bands were polymorphic. Genetic Jaccard's similarity coefficient was estimated in the range of 0.15–0.63, and with a mean of 0.35. Results showed that the genetic diversity was not according to the geographical diversity.     

Identification of parental species of the Alstroemeria cv. ‘Jubilee’ using AFLP marker technique

Twelve Alstroemeria species, two hybrids, one cv. ‘Jubilee’, an anther-cultured plant from cultivar ‘Jubilee,’ and Bomarea salsilla and Leontochir ovallei (the latter two were chosen as outgroup) were evaluated using the AFLP marker technique in order to identify putative parental genotypes of the Alstroemeria cv. ‘Jubilee’ and of known interspecific hybrids, and to estimate their genetic relationships within the genus Alstroemeria. A total of 297 AFLP markers were scored by using the primer combination (E + ACCA/M + CTAG). In order to discriminate all Alstroemeria genotypes, cluster analysis (UPGMA) and principal coordinates analysis were performed. The Alstroemeria cv. ‘Jubilee’, of which the parents are unknown, had genetic distance (GD) 0.54 from Alstroemeria exserens, GD 0.57 from Alstroemeria garaventae, GD 0.62 from Alstroemeria gayana, and GD 0.66 from Alstroemeria hookeri cumminghiana. Thus, these four species are considered as putative parental genotypes. An interspecific hybrid (Alstroemeria aurea × Alstroemeria inodora), showed the smallest genetic distance from A. aurea (GD 0.56) and A. inodora (GD 0.45). The Alstroemeria ligtu group was distantly allocated from other Chilean species. We conclude that the AFLP marker technique appears to be a satisfactory tool for identifying the parental genotypes of interspecific hybrids in Alstroemeria.     

Genetic diversity and population structure of an endangered Orchid (Dendrobium loddigesii Rolfe) from China revealed by SRAP markers

Dendrobium loddigesii Rolfe is an endangered perennial herb with ornamental and medicinal value. Due to habitat deterioration and human over-exploitation, it has suffered a significant decline in abundance. Determining the level of genetic diversity and pattern of population genetic structure of this species would be helpful for its conservation and management. In this paper, sequence-related amplified polymorphism (SRAP) markers were applied to assess the level and pattern of genetic diversity in seven populations of D. loddigesii. Seventeen SRAP primer combinations generated a total of 231 clear amplification bands encompassing 187 (80.95%) polymorphic bands. A high level of genetic diversity was detected (PPB = 80.52%, H = 0.2743, I = 0.4113) at the species level. There was a moderate genetic differentiation (G_st = 0.304) among populations. Two main clusters were detected by cluster analysis using the unweighted pair-group method with arithmetic average (UPGMA). Mantel test revealed that no significant positive correlation was found between genetic distances and geographic distances (r = 0.2302; P > 0.05). Recommendations for conservation of the endangered species resources are proposed.     

Characterization of a new fungal antagonist of Phytophthora capsici

The capacity of many microorganisms for antagonism towards pathogens is unknown and previously uncharacterized species may be potentially useful in this respect. This work presents the evaluation of the capacity of a previously uncharacterized Ascomycete, denominated here as UA to protect chili pepper (Capsicum annuum) against Phytophthora capsici, and other soil-borne plant pathogens and establishes the role of UA as a biocontrol agent. Inoculation of UA 2 days before the inoculation of P. capsici led to 77.8% survival of pepper plants. Simultaneous confrontation of both microorganisms in vitro led to 53.1% growth inhibition of P. capsici, while the inoculation of P. capsici 3 days after the inoculation with UA improved growth inhibition up to 73%. Simultaneous confrontation in vitro of UA with Fusarium oxysporum and Fusarium solani led to 41.2 and 50% growth inhibition, respectively, but had no effect on Rhizoctonia solani or a binucleate Rhizoctonia isolate. Moreover, formation of zoosporangia and the germination of zoospores were completely inhibited by exposure to undiluted filter sterilized filtrate. UA produces septate mycelia, but could not be classified in detail due to a lack of spores or reproductive structures. However, sequencing of Internal Transcribed Spacer 1, 2 and the 5.8S genes indicated that this fungus is a member of the Ascomycetes.     

Genetic divergence among inbred lines in Cucurbita moschata from China

Knowledge of genetic divergence among inbred lines is essential for cross breeding. The objectives of this study were to (1) analyze the level and character of genetic diversity in C. moschata accessions, and (2) classify the genetic divergence among inbred lines in C. moschata to assist in selection of parent genotypes for genetic improvement. Twenty agronomic characters were investigated and rich diversities were demonstrated among 39 inbred lines of C. moschata from China. Various degree correlations among these characters made it possible to summarize the diversities of the twenty characters into 3 major principal components: leaf, fruit and flesh quality factor. Forty-one inbred lines of pumpkin were clustered into four groups based on principal component data, which is more distinct for classification that based on the original data of the 20 characters. However, parent inbred lines whose hybrids displayed significant heterosis in fruit weight, soluble solid and fruit shape were located in different clusters or sub-clusters based on standardized original data. It was suggested that genotypes in the same clusters may represent members of one heterotic group.     

Development of ITS sequence based SCAR markers for discrimination of Paphiopedilum armeniacum, Paphiopedilum micranthum, Paphiopedilum delenatii and their hybrids

Paphiopedilum armeniacum, Paphiopedilum micranthum and Paphiopedilum delenatii are endangered orchid species. These three Paphiopedilum species and their hybrids are difficult to distinguish morphologically. In this study, rDNA-ITS (internal transcribed spacer) sequences were used to design species-specific SCAR (sequence characterized amplified regions) markers to distinguish P. armeniacum, P. micranthum, P. delenatii and their respective hybrids. The developed markers efficiently amplified 600 bp DNA product for P. armeniacum and its hybrids (SCAR-600armF/Pap-ITS2R), 300 bp product for P. delenatii and its hybrids (SCAR-300delF/Pap-ITS2R) and 700 bp product for P. micranthum and its hybrids (SCAR-700micF/Pap-ITS2R). The effectiveness of designed species-specific markers was also confirmed by using multiplex polymerase chain reaction amplification with a combination of developed three SCAR markers.     

Genetic characterization of different demes in Prunus persica revealed by RAPD markers

DNAs of 180 accessions in 10 demes in Prunus persica were amplified with twenty-two, 10-base primers selected from 200 arbitrary primers using Randomly Amplified Polymorphic DNA (RAPD) technology. One hundred and eighty loci were observed and recorded. With statistical analyses of the data from the study, genetic diversity of the demes was expressed as follow: yellow peach group > honey peach group > flat peach group > red leaf peach group > crisp peach group > bitao group and juicy peach group > nectarine group > shouxingtao group > weeping peach group. Genetic variations among and within groups by AMOVA analyses were 11.9, 88.1%, respectively. Demes clustered by UPGMA modified from NEIGHBOR procedure of PHYLIP Version 3.5, the edible peaches of which were combined as a section, while the ornamental species were classified into separate sections. Through analyses of genetic diversity and genetic structure, the results could provide molecular biological evidence for conservation and utilization of P. persica germplasm.     

Identification of seven haplotype-specific SFBs in European pear (Pyrus communis) and their use as molecular markers

The European pear (Pyrus communis) carries the S-RNase-mediated gametophytic self-incompatibility (GSI) system. The S-haplotype is conferred by an S-locus, which contains the style-specific expressed S-RNase, and the pollen-specific expressed F-box genes (SFB). Both the S-RNase and the SFB genes are multi-allelic and each is characteristic of one of the S-haplotypes. Therefore, they are ideal markers for molecular S-genotyping. In this work, for the first time, seven haplotype-specific SFBs were isolated from European pears. Particular primers for each of these SFBs were generated, thus providing an additional tool for S-genotyping of European pear cultivars.     

Genetic characterization of Allium tuncelianum: An endemic edible Allium species with garlic odor

Allium tuncelianum (Kollman) Özhatay, Matthew & ?iraneci is a native species to the Eastern Anatolia. Its plant architecture resembles garlic (Allium sativum L.) and it has mild garlic odor and flavor. Because of these similarities between two species, A. tuncelianum has been locally called “garlic”. In addition, both A. tuncelianum and garlic has 16 chromosomes in their diploid genomes. Recently, A. tuncelianum has been suggested as the wild progenitor species of garlic. In this study, amplified fragment length polymorphisms (AFLP) markers and nucleotide sequence analysis of the internal transcribed spacer region (ITS) were used to assess genetic and phylogenetic relationships among A. tuncelianum, garlic and some other Allium species. AFLP analysis demonstrated that A. tuncelianum and garlic are genetically distinct and they are likely different species. Phylogenetic analyses based on the nucleotide sequence of ITS suggested that A. tuncelianum and garlic are distinct species and placed A. tuncelianum, garlic, Allium ampeloprasum and Allium scorodoprasum into the same clade in the neighbor joining dendrogram and in the consensus tree of parsimony analysis. However, A. tuncelianum was phylogenetically less related to garlic than either A. ampeloprasum or A. scorodoprasum, suggesting that A. tuncelianum may not be the immediate wild ancestor species of garlic. Further studies to generate hybrid progeny between A. tuncelianum and garlic (if possible) could provide more information on the homology between the chromosomes of A. tuncelianum and garlic and genetic relationships between these two species.     

Genetic diversity of bitter gourd (Momordica charantia L.) genotypes revealed by RAPD markers and agronomic traits

Bitter gourd or bitter melon (Momordica charantia L.) is considered as minor cucurbitaceous vegetable in spite of having considerable nutritional and medicinal properties. Although some reports on genetic diversity based on morphological characterization are available, no work has been conducted to estimate genetic diversity using molecular markers in this crop. In the present study, 38 genotypes of M. charantia including few commercially cultivars collected from different parts of India based on agro-ecological zones were analysed for diversity study both at morphological and molecular levels. Genomic DNA was extracted from young healthy leaves following the procedure of Doyle and Doyle [Doyle, J.J., Doyle, J.L., 1990. A rapid DNA isolation procedure from small quantity of fresh leaf material. Phytochem. Bull. 119, 11–15]. Pair-wise comparison of genotypes was calculated as per the procedure of Jaccard [Jaccard, P., 1908. Nouvelles recherches sur la distribution florale. Bull. Soc. Vaud. Sci. Nat. 44, 223–270]. Dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA) and the computation for multivariate analysis was done using the computer programme NTSYS-pc Version 2.0 [Rohlf, F.J., 1998. NTSYS-pc Numerical Taxonomy and Multivariate Analysis System, Version 2.01. Exeter Software, Setauket, NY, USA]. Diversity based on yield related traits and molecular analysis was not in consonance with ecological distribution. Among 116 random decamer primers screened 29 were polymorphic and informative enough to analyse these genotypes. A total of 208 markers generated of which 76 (36.50%) were polymorphic and the number of bands per primer was 7.17 out of them 2.62 were polymorphic. Pair-wise genetic distance (GD) based on molecular analysis ranged from 0.07 to 0.50 suggesting a wide genetic base for the genotypes. The clustering pattern based on yield related traits and molecular variation was different.     

Assessment of diversity in Podophyllum hexandrum by genetic and phytochemical markers

For successful conservation and domestication of a species, evaluation of its genetic diversity by different markers is important. Morphological characteristics, phytochemical variation and random amplified polymorphic DNA (RAPD) profiles were generated in different accessions of Podophyllum hexandrum in order to determine the genetic diversity. Random amplified polymorphic DNA (RAPD) analysis revealed a high degree of genetic diversity among the accessions used in the study. There was also high diversity in the concentration of marker compounds in the collected samples as revealed by HPLC analysis. It is shown that the approaches used in the work successfully discriminate between the accessions of this species and thus they constitute interesting tools to analyze molecular, biochemical and phenotypic diversity within this species. Similarity measurement using UPGMA followed by cluster analysis resulted in formation of many groups based on geographical distribution that generally reflected expected trends between the genotypes. There were also some important exceptions like PW-S, an accession from Wastoorwan, Khrew showing close resemblance to PG-S and PG-B collected from Gulmarg but grown at two different gene banks at Srinagar and Bonera. Further an accession PSH-B from Keller was significantly diverse from the rest of the native genotypes phytochemically, morphologically and at molecular level. RAPD data analysis was found to be significant predictor of phytochemical markers in cultivated P. hexandrum germplasm. Twelve accessions grown in gene bank repository were subjected to RAPD analysis and were assessed for content of podophyllotoxin and podophyllotoxin β-d-glycoside by HPLC. Individual regressions of podophyllotoxin and podophyllotoxin β-d-glycoside by RAPD analysis against HPLC has been found to determine linear values. Strong correlation and a strong association of values of the phytochemical variables and the DNA polymorphism data has been recorded.     

Analysis of phenetic relationship between Citrus indica Tanaka and a few commercially important citrus species by ISSR markers

Analysis of polymorphism in ISSR amplicons revealed diverse genetic relationship between Citrus indica and five other citrus species. In a consensus UPGMA dendrogram, based on Nei and Li's distance matrices, the C. indica samples from three different sites of Meghalaya, India were clustered together with 99% bootstrap support. C. reticulata, C. sinensis, C. aurantifolia formed a cluster with 67% bootstrap separation. C. macroptera and C. maxima samples from two sites of Meghalaya formed separate clusters with respectively 100% and 98% bootstrap supports. Principal Components Analysis projected C. indica to be more closely related to C. aurantifolia than to the other citrus species of the study. PCA also exposed variations within the C. maxima and C. macroptera samples.     

Molecular characterization and analysis of somaclonal variation in chrysanthemum cultivars using RAPD markers

Molecular characterization using RAPD analysis was carried out in eight cut flowers and two pot plant cultivars of chrysanthemum. Three of them (‘Refocus’, ‘Red Reagan’, and ‘Sheena Select’) were established in vitro and the occurrence of somaclonal variation was studied using the same molecular technique. Two induction media (MS + 0.1 mg l⁻¹ NAA + 0.1 mg l⁻¹ BA, and MS + 2.0 mg l⁻¹ IAA + 0.5 mg l⁻¹ Kinetin), and two proliferation media (MS + 0.1 mg l⁻¹ NAA + 0.2 mg l⁻¹ BA, and MS + 4.0 mg l⁻¹ IAA + 2.0 mg l⁻¹ Kinetin) were employed in order to evaluate the effect of the medium composition in the shoots’ stability. Likewise, the effect of the culture age was considered in assessing genetic stability. Monthly subcultures were carried out, identifying the origin and history of the shoots, throughout a nine-month proliferation period followed by acclimatization. Molecular markers were obtained in every subculture cycle and from the acclimatized plants. Only one shoot from the 7th subculture of the cultivar ‘Refocus’ showed a different band pattern. The use of RAPD for chrysanthemum cultivar characterization and somaclonal variation detection is discussed.     

Development of microsatellite markers in cultivated vanilla: Polymorphism and transferability to other vanilla species

The sources of natural vanilla are the cured fruits of two obligatorily hand-pollinated and clonally propagated orchids: ‘Bourbon/Mexican vanilla’ (Vanilla planifolia G. Jackson) and ‘Tahitian vanilla’ (Vanilla tahitensis J.W. Moore). In this paper we describe for the first time the isolation and characterization of 14 microsatellite loci from V. planifolia. These were monomorphic within cultivated accessions, as expected from the probable single clonal origin of this crop and previous genetic studies. These markers were transferable to V. tahitensis and 11 loci were polymorphic between these two closely related species. Furthermore, some of these markers were transferable and polymorphic across 15 other wild American, African and Asian species and revealed consistent relationships between species, together with a strong pattern of Old World versus New World differentiation in the genus. These microsatellites will be very useful for diversity, hybridization and phylogeographic studies in the genus Vanilla.     

EST-PCR markers developed for highbush blueberry are also useful for genetic fingerprinting and relationship studies in rabbiteye blueberry

The pedigrees of most rabbiteye blueberry (Vaccinium virgatum) cultivars can be traced back to four wild selections, ‘Ethel’, ‘Clara’, ‘Myers’, and ‘Black Giant’; thus, they result from a very narrow germplasm base and are highly related. Until now randomly amplified polymorphic DNA (RAPD) has been the only type of molecular marker used in rabbiteye blueberry. Here we have tested whether a type of sequence-tagged site (STS) marker which utilizes specific ∼20-mer primers from expressed sequence tags (ESTs) of highbush blueberry (V. corymbosum), called EST-PCR markers, are useful for genetic fingerprinting and relationship studies in rabbiteye blueberry. Of 44 EST-PCR primer pairs, from an assortment of genes expressed in flower buds of cold acclimated and non-acclimated plants, and shown to amplify polymorphic fragments among a collection of highbush genotypes, 40 (91%) resulted in successful amplification, and 33 of those (83%) amplified polymorphic fragments among the rabbiteye genotypes. The average number of scorable bands per primer pair was two. A dendrogram constructed from genetic similarity values, based on the EST-PCR marker data, tended to group siblings and parent/progeny together, generally agreeing with pedigree information. A group of 20 markers from five EST-PCR primer pairs distinguished all the genotypes in this study. These markers are as easy to generate and as affordable as RAPDs, but are based on actual gene sequences, and should have general utility for DNA fingerprinting, genetic diversity, and mapping studies.     

Rose (Rosa hybrida L.) EST-derived microsatellite markers and their transferability to strawberry (Fragaria spp.)

The ‘Genome database for Rosaceae (GDR)’ provides a large collection of expressed sequence tags (ESTs) harboring simple sequence repeats (SSRs) from several Rosaceae genera, including Rosa (rose). Primer pairs flanking SSR were designed for 312 unique Rosa ESTs based on GDR database. Eight rose (Rosa hybrida L.) genotypes were tested for PCR amplification, and 287 (92%) of the primer pairs generated allele-specific PCR bands that were readily scored. From 183 (63.7%) primer pairs that evidenced polymorphic alleles among the eight rose cultivars, 20 pairs evidencing EST sequence homology to known gene functions and high levels of polymorphism were selected and utilized for DNA fingerprinting and genetic diversity assessments of 47 rose hybrids. A total of 202 polymorphic bands were scored and generated unique fingerprints for each rose hybrid. The Nei–Li genetic similarity coefficients among 1081 pair-wise comparisons of 47 cultivars exhibited a broad range of genetic variations from 0.30 (‘Grand King’ and ‘Carnival’) to 0.99 (‘First Red’ and ‘Red Champ’). UPGMA cluster analysis divided 47 hybrids into five major groups and two sub-groups. The cross-species transferability of 273 EST-SSR primer pairs was evaluated using four genotypes of the strawberry, a genus member of the Rosaceae family. PCRs on the DNA samples of strawberry were successful for 165 primer pairs; among these, 123 pairs amplified 243 polymorphic bands. As surrogates of the marker transfer, the phenetic relationship among the four strawberry genotypes was evaluated. Genetic similarity coefficients varied from 0.78 (‘Maehyang’ and ‘Janghyee’) to 0.64 (‘Janghyee’ and ‘Pragana’). The results of cluster analysis showed that the three octaploid strawberry cultivars were quite similar, whereas the diploid ‘Pragana’ was related distantly at the genomic DNA level. The EST-SSR markers developed in the present study can be efficiently utilized for genetic diversity studies in Rosaceae.