Evaluation of genetic diversity among some Iranian wild asparagus populations using morphological characteristics and RAPD markers

An evaluation of genetic diversity in 39 wild asparagus populations was carried out using morphological and RAPD markers. A combination of morphological traits and random RAPD primers was used to examine the level of genetic variation and polymorphisms among the populations. A factor analysis using Ward's method on mean values of morphological characteristics indicated seven main factors resulting in four groups. Analysis of polymorphic bands using Jaccard's similarity coefficient indicated that genetic similarity ranged between 0.71 and 0.29. At a similarity level of 0.64, the populations were divided in three sub-clusters, containing 34, four and one populations, respectively. Significant regression associations were found between 21 morphological characteristics and 18 RAPD markers, revealing some informative markers associated with some traits. The highest R² was related to 18 RAPD markers associated with gender (53.5%) that among them BA-04₂₀₀₀ had a maximum R². The results showed that Iranian wild asparagus with its high levels of genetic variation could be considered as a valuable gene pool for future asparagus breeding programs. Furthermore, it could be inferred that morphological characteristics and RAPD markers are suitable tools to discriminate asparagus populations for the evaluation of genetic diversity.     

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.     

Characterization of pea accessions by SRAP's markers

Sequence-related amplified polymorphism (SRAP) and morphological markers to estimate the genetic relations among forty pea varieties (Pisum sativum L.) were studied. Data on 15 morphological traits were collected and analyzed. A total of 162 polymorphic SRAP's bands were scored using seven combinations of primers. Cluster analysis and both principal component and principal coordinate analysis were carried out. The varieties were grouped in four clusters through procrustes generalized analysis. Relationships among varieties revealed by molecular markers were significantly correlated with those based on the agronomic traits, suggesting that the two systems give similar estimates of genetic relations among the varieties. Parents selection depend on the specific objectives in further breeding programs.     

Genetic structure of Buxus sinica var. parvifolia, a rare and endangered plant

Buxus sinica var. parvifolia, a rare and endangered tree species in some semitropics alpine areas of China, plays an important role in the maintenance of the landscape and ecosystem. In this study, RAPD and ISSR markers were used to investigate the genetic diversity and structure of five natural populations and one tamed population of B. sinica var. parvifolia. 21 RAPD primers amplified 209 bands with 167 (79.90%) polymorphic and 21 ISSR primers amplified 518 bands with 467 (90.15%) polymorphic. The genetic diversity, estimated by Shannon’ index, was 0.4343 (by RAPDs) and 0.3661 (by ISSRs). Both RAPD and ISSR analyses revealed a high level of genetic diversity in natural populations of B. sinica var. parvifolia. Furthermore, analysis of molecular variance (AMOVA) was used to apportion the variation within and between populations. The proportion of variation attributable to within-population differences was very high (69.2% by RAPDs; 84.51% by ISSRs). Moderate differentiation was detected among populations using RAPDs (30.80%), while only a small amount of variation (15.49%) was detected among populations using ISSRs. We suggest that the present genetic structure is due to high levels of environmental variability and gene flow, which still need further study to confirm. Conservation measures are suggested, including in situ and ex situ strategies, based on the observed population genetic information.     

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.     

Fingerprinting of three typical macrosperma Italian lentil (Lens culinaris Medik.) landraces using fluorescence-based AFLP markers

Italian lentil landraces are principally cultivated for self or local consumption. Most of them are disappearing, particularly macrosperma types by being less required by the market. A pre-requisite for the conservation and the efficient use of genetic resources is the better understanding of the extent and the distribution of the existing genetic variation, useful for future breeding programmes. Our study was undertaken to analyse and quantify the genetic diversity within and among three macrosperma Italian lentil landraces (Onano, Altamura and Villalba), using fluorescent AFLP markers. AFLP markers generated information to differentiate among closely related genotypes and group within the same cluster individuals belonging to the same landrace. The total genetic diversity (H_T), the genetic diversity within population (H_S) and the extent of differentiation between populations (D_ST) were 0.198, 0.155 and 0.043, respectively. The fixation index (G_ST = 0.219) showed that about 78% of the observed total genetic variation can be attributed to within population differences and around 22% is due to differences among populations. The gene flow estimate (N_m = 1.774) and the mean genetic distance value (0.077) suggested narrow genetic base among the analysed populations, confirming the tendency of Italian lentil landraces to group together. The present study showed that fluorescence-based AFLP technique is a biotechnological tool that can provide significant insights for research in genetic diversity of lentil landraces and their subsequent conservation and utilization in breeding programs.     

Genetic diversity of ash gourd [Benincasa hispida (Thunb.) Cogn.] inbred lines based on RAPD and ISSR markers and their hybrid performance

Ten inbred lines of ash gourd [Benincasa hispida (Thunb.) Cogn.] were crossed to produce 45 F₁ hybrids (without reciprocal) which were evaluated along with the parents for 20 growth- and yield-related traits, in a replicated field trial. High level of heterosis was observed among the hybrids for most of the traits examined, including yield. These inbred lines were analysed by using 42 RAPD primers those produced 282 DNA marker bands. A total of 130 RAPD markers were obtained with a mean of 3.1 per primer, which in combination discriminated all the inbreds from each other. Pair-wise genetic distance measurements ranged from 0.07 to 0.31, suggesting a wide genetic diversity for these inbreds. These inberds were also analysed with five ISSR primers of which four were informative. Twenty-six ISSR marker bands were generated of which 11 were polymorphic with an average of 2.80 per primers. The percentage of polymorphic bands produced were higher in ISSR markers (>80%) than generated through RAPD markers (46%). Although the results indicated significant positive correlations of genetic distance with hybrid performance and heterosis, the RAPD based genetic distance measures and use of limited ISSR markers in this present study could not effectively predict hybrid performance in this crop. The genetic variation among ash gourd inbred lines examined, herein, defined a marker array (combined ISSR and RAPD) for the development of a standard reference for further genetic analyses, and the selection of potential parents for predicting hybrid performance and heterosis.     

Genome composition and genetic diversity of Musa germplasm from China revealed by PCR-RFLP and SSR markers

Banana (Musa spp.) is one of the most important crops in the world. In this study, 216 banana accessions, 184 from the National Banana Germplasm Collection of China (NBGCC) and 32 from the International Network for the Improvement of Banana and Plantain (INIBAP), were used to determine the genome composition of banana plants in these collections and to estimate their genetic diversity. The genome composition was examined using PCR-RFLP markers. The molecular data for all but one accession (ITC 1231) from INIBAP were in agreement with the initial records based on phenotypic characteristics. Microsatellite (SSR) markers were used to investigate the genetic variability and relationships among these banana accessions. Ten of the 47 primer pairs tested consistently produced reproducible and discrete fragments. We identified a total of 92 alleles, ranging from 5 to 15 per locus. The genetic similarity between the accessions ranged between 0.1 and 1, when estimated using Jaccard's coefficient. The UPGMA method based on genetic similarities, grouped the NBGCC accessions according to those containing the ‘A’ and ‘B’ genomes. However, this analysis could not separate all the accessions, especially the somatic mutations, using the primers in this study. These data indicated that limited genetic variation exists within these accessions and the collections of NBGCC should include a much wider range of banana plant material.     

Pericarp total protein profiles as molecular markers of tomato fruit quality traits in two segregating populations

The tomato fruit quality results of biochemical and physiological changes that occur during the ripening process. Although, the pericarp total protein profiles are less polymorphic than DNA-based markers the polymorphism in those could be directly associated with fruit quality traits. The aim of this work was to identify associations between polymorphic polypeptides from fruit pericarp at two ripening stages and fruit quality traits evaluated in two segregating populations of tomato. A cross between a normal ripening cultivar of Solanum lycopersicum (C, Caimanta) and a genotype carrying the nor (non ripening) gene (N) as well as a cross between Caimanta and a cherry type tomato of S. lycopersicum var. cerasiforme (Ce) showed genetic variance for several fruit quality traits such as fruit weight, shape, solids soluble content, acidity, color and fruit shelf life. The quantitative variations observed at phenotypic level had correspondence with the polymorphism detected in the protein profiles. Indeed, the polymophic polypeptides associated with quality fruit traits and fruit shelf life would be useful to assist tomato breeding programs as protein molecular markers.     

Molecular polymorphism of cytoplasmic DNA in Ficus carica L.: Insights from non-coding regions of chloroplast DNA

The trnL (UAA) intron and the intergenic spacer between the 3′ exon of trnL (UAA) and trnF (GAA) sequences were used as genetic markers for differentiating Ficus carica cultivars and establishing refined genetic relationships. The study was based on 20 fig cultivars, collected from south and centre of Tunisia. Since, the intron was thought to be more variable among close relatives than is the chloroplast spacer. The size of these non-coding regions varied from 554 to 589 and from 989 to 1022 bases pairs for the intron and the combined sequences correspondingly. The average of GC content was 33.9% and 34.6% in the intron and the combined intron and spacer respectively. High values of A + T contents were detected in both data sets and may explain the high proportions of transversions founded. The observed variation pattern of plastid DNA provides evidence of an important genetic diversity. The overall transition/transversion bias (R) was 0.202 in the intron and 0.27 in the combined regions. The RI index of 0.592 indicates that these combined sequences have clearly more homoplasy then the intron (RI = 0.705) and spacer (RI = 0.777) sequences separately. Phylogenetic trees were generated based on maximum parsimony (MP) and neighbor-joining (NJ) analysis of the chloroplast sequences data. Results proved that a typically continuous genetic diversity characterizes the local fig germplasm. In fact, relationships inferred from the cpDNA analysis suggest several clades, which do not show geographical or tree sex correspondence. Although the level of apparent diversity is considerable, we may conclude that non-coding regions of chloroplast genome provide a new and practical opportunity to evaluate genetic diversity and to discriminate fig cultivars. Revealed cytoplasmic DNA markers are reliable to elaborate a molecular data base to conduct management and breeding programs on local fig germplasm.     

Genetic diversity of Dimocarpus longan in China revealed by AFLP markers and partial rbcL gene sequences

Amplified fragment length polymorphism (AFLP) and partial rbcL gene sequencing were used to investigate genetic diversity among various longan (Dimocarpus longan Lour) accessions as well as a presumed closely related species Dimocarpus confinis How et Ho and litchi (Litchi chinensis Sonn). No significantly shared AFLP fragment was found between the three species, indicating that D. confinis and litchi are very far in genetic distance from any longan accession studied. Partial rbcL sequences of 501 bp from the first coding site in these species were obtained, which revealed several substitutes. One such DNA base pair substitute resulted in an amino acid difference between longan and litchi. Furthermore, another 4 bp resulted in a two amino acid difference between longan and D. confinis, which was consistent with AFLP results and indicated that D. confinis should be excluded from the longan genus, Dimocarpus. Within the longan species, no DNA substitute was found. Using nine primer combinations, a total of 66 AFLP markers were obtained from 41 longan accessions. One non-Chinese longan accession ‘Miaoqiao’ was distinctly different from all other longan cultivars collected in China, indicating that more genetic resources of longan might be collected also from longan production regions outside of China. AFLP markers might be developed to identify longan cultivars as well as expedite progeny screening in breeding programs of this perennial fruit tree.     

Germplasm diversity and genetic relationships among walnut (Juglans regia L.) cultivars and Greek local selections revealed by Inter-Simple Sequence Repeat (ISSR) markers

Inter-Simple Sequence Repeat (ISSR) markers were used for the assessment of genetic diversity among walnut (Juglans regia L.) selections from Greek native populations in comparison to internationally cultivated walnut genotypes. Similarity coefficient values from 0.13 to 0.93 (with an average of 0.48) were found among the 56 accessions examined, which indicated the presence of a high degree of genetic variability. Most international cultivars were grouped together while most Greek native populations could not be placed into a distinct group. The Greek native population genotypes were found more diverse than the international cultivars. The mean similarity coefficient values for the former and latter were 0.44 and 0.56, respectively. In the cultivar group, two subgroups were distinguished; one consisted of genotypes involving ‘Payne’ and the other ‘Franquette’ in their pedigrees. Some cultivars and populations could not be grouped according to their pedigrees or collection area. Analysis of molecular variance (AMOVA) revealed that a larger part of the genetic variation exists among Greek walnut populations within a collection region (89%) than among the regions (11%). The pairwise regional PhiPT values indicated that the most geographically distant regions are the most genetically differentiated. The high variability existing in the Greek germplasm in combination with their valuable agro-morphological traits suggested that it would be beneficial to utilize this native germplasm pool in walnut breeding programs and germplasm management activities to maximize genetic diversity in cultivated walnut.     

Amplified fragment length polymorphism (AFLP) markers for fingerprinting of Argyranthemum frutescens cultivars

Argyranthemum frutescens is a commercially important ornamental species with extensive breeding programmes in several countries. Since it is vegetatively propagated there is a great need for tools for identification of cultivars. Vegetatively propagated species require clean-up from diseases, often performed through meristem-tip cultures. Forty-three cultivars of A. frutescens propagated by meristem-tip culture and traditional cuttings were analyzed for genetic relatedness and possible somaclonal variation using AFLP. Five primer combinations resulted in a relatively high degree of polymorphism. Ten molecular markers generated by one primer combination could distinguish between all 43 cultivars. Differences in fingerprints between meristem-tip culture and cuttings from the same cultivars were found, but the proportion of fragments being specific for either tissue culture or cuttings was relatively low. Some cultivars that did not display somaclonal variation as judged by the AFLP-fingerprints may still be genetically unstable since some morphological variation was observed in the true to type test.     

Development of DNA markers for hybrid identification in Leucadendron (proteaceae)

A rapid and reliable method to accurately identify hybrids at an early age is essential to the success of Leucadendron breeding programs because identification based on morphology can be difficult or impossible when the seedlings are young. DNA based PCR-RFLP and random amplified microsatellite polymorphism (RAMP) markers were developed for this purpose. Unexpected non-parental fragments appeared during the PCR-RFLP analysis of the nuclear ITS region of L. uliginosum 05 × L. procerum 04 hybrids. Mixing DNA from both parents in a single PCR also produced the non-parental fragment, suggesting that PCR recombination had introduced a novel restriction site into the products from the hybrids. Sequencing of individual amplified ITS products from the hybrids confirmed this conclusion. To avoid this complication, RAMP markers were developed for accurate hybrid identification in Leucadendron. RAMP analysis generated a considerable number of polymorphic products, and showed more discrimination in identifying Leucadendron hybrids than did PCR-RFLP.     

Application of simple sequence repeat (SSR) markers in apricot breeding: molecular characterization,protection, and genetic relationships

Seventeen peach simple sequence repeat (SSR) markers have been used in the molecular characterization of 8 apricot (Prunus armeniaca L.) cultivars from Spain, North America, France, and Greece; a new breeding line from the apricot breeding program of INRA (Avignon, France); and 13 breeding lines and 3 new releases from the apricot breeding program of CEBAS-CSIC (Murcia, Spain). DNA fingerprints have been developed establishing the genetic relatedness among cultivars, new releases, and breeding lines. Amplification of SSR loci was obtained for all 17 primer pairs and 14 of them produced polymorphic amplification. The number of presumed alleles revealed by the SSR analysis ranged from one to nine, although most primers revealed three alleles or more. The mean number of alleles per locus was 4.1. Results allowed the molecular identification of all the apricot genotypes assayed. Apricot genotypes clustered into seven principal groups in accordance with their origin and pedigree. The implications of these results for apricot breeding programs in terms of protection of new release and design of new crosses are also discussed.     

Development of novel chloroplast microsatellite markers for Dendrobium officinale, and cross-amplification in other Dendrobium species (Orchidaceae)

Nine pairs of polymorphic chloroplast microsatellite primers were developed for Dendrobium officinale Kimura et Migo, an endangered herb. Levels of polymorphism were tested across a total of 55 individuals from four natural populations (12–15 individuals per population). Allele numbers varied from two to four per locus, while the number of haplotypes ranged from four to six per population. Transferability of the nine polymorphic chloroplast microsatellite primers was checked on an additional set of 51 Dendrobium individuals (belonging to 17 different species). Three markers could be transferable to all the species tested, while the remaining six markers successfully cross-amplified in most species tested. Moreover, polymorphism of the nine chloroplast microsatellite primers was tested across Dendrobium moniliforme (L.) Sw. and Dendrobium loddigesii Rolfe. All of them were polymorphic in D. moniliforme, while seven of which were polymorphic in D. loddigesii. These polymorphic chloroplast microsatellite primers developed for D. officinale will be a useful tool for the study of genetic diversity, population genetic structure, evolution of D. officinale and establishment of effective conservation strategies.     

Genetic variability in wild vs. cultivated Eruca vesicaria populations as assessed by morphological,agronomical and molecular analyses

In this study, the genetic diversity of 50 individuals of rocket, Eruca vesicaria, from five accessions, four of them wild type collected from different parts of Spain and one commercial, were evaluated using morphological, agronomical and inter simple sequence repeat DNA (ISSR) data. Molecular analysis was carried out using the ISSR technique with 20 primers. Out of these 20 primers, nine were polymorphic, producing a total of 395 DNA bands, 247 of which were polymorphic among the accessions. A dendrogram drawn on the basis of a similarity matrix using the UPGMA algorithm revealed that the 50 samples of rocket plants could be classified into three major clusters at a Nei's genetic distance of 0.36. The experiment shows that molecular markers such as ISSR are a good instrument for distinguishing and selecting rocket accessions to group different wild populations. In general, a high variation was observed for most of the 16 morphological and 6 agronomical traits showing significant differences. Some morphological traits such as leaf length, petiole length and lamina width explained 69.1% of the whole variation observed in the populations, and some agronomical traits such as leaf area, nitrate and chlorophyll contents accounted for 65.7%, but the clusters generated by means of agronomical and morphological variables were less evident than when ISSR markers used. Some accessions showed good qualities, such as small leaves, high chlorophyll content, late-flowering or low nitrate content. All these parameters, together with the high degree of genetic homogeneity found, could make the local accessions good candidates for a future breeding programme.     

Genetic diversity and population's structure in Tunisian strawberry tree (Arbutus unedo L.)

A growing interest in the use of the Strawberry tree (Arbutus unedo L., Ericaceae) has been recently reported for industrial, pharmaceutical and chemical fields. However, the bulk material comes from natural populations because of the lack of selection of interesting cultivars. In Tunisia, A. unedo populations are severely destroyed due to deforestation and over-collecting. The species occurs in small scattered populations decreasing progressively in size. Yet, no conservation or improvement programs are attempted to preserve and promote the potential value of this resource. In this work, we assessed the genetic diversity of nine Tunisian populations of A. unedo L. from different bioclimates, using 65 polymorphic RAPD loci. The analysis of the genetic variation within and among populations is primordial to elaborate conservation and improvement programs. A low genetic diversity within a population estimated by both Nei's (He) and Shannon's diversity (H′) indices (0.155 < He < 0.248; 0.229 < H′ < 0.364) was observed due to genetic drift and selfing. At the species level, the amount of the within population variation estimated by Shannon's index (H_POP/H_SP = 0.686) and the molecular variance (80.67%) was higher than that among populations. A moderate genetic differentiation among populations (Φ_ST = 0.193 and G_ST = 0.314) which could be attributed to the long seed distance dispersal was detected. The UPGMA dendrogram based on Φ_ST values showed three clusters each including populations without relationship to bioclimatic or geographical origin indicating that differentiation occurs at a local space scale. The in situ protection measures should be made appropriately according to a population within bioclimates. The ex situ conservation and the selection of genotypes should involve extensive collection of seeds or cuttings from the within populations rather than among them.     

Genetic structure of mulberry from different ecotypes revealed by ISSRs in China: An implications for conservation of local mulberry varieties

Mulberry is a perennial and economically important plant that has traditionally been used for feeding the silkworm. Evaluating genetic relationship is important for long-term improvement in mulberry yield, quality and resistance, and for germplasm conservation and identification. Population structure and genetic diversity of 8 mulberry populations from different ecotypes in China were analyzed by ISSR markers. Twelve ISSR primers generated a total of 83 amplification products, of which 50 were polymorphic, revealing 60.24% polymorphism among 66 mulberry local varieties, the mean PIC value was 0.1469. The total heterozygosity (H_T), heterozygosity within population (H_S), diversity between populations (D_ST) were 0.1600, 0.0851 and 0.0749, respectively. The coefficient of population differentiation (G_ST) was 0.4683, indicating that the variations among populations and those within populations contributed 46.8% and 53.2% to the total heterozygosity, respectively. The gene flow (N_m) was 0.5678, suggesting that genetic drift between populations can caused local genetic differentiation and therefore, population divergence. The mean genetic similarity coefficient was 0.8456, genetic similarity coefficient among 8 mulberry populations ranged from 0.8441 to 0.9640, indicating that genetic diversity of different populations existed variation. A dendrogram of all 66 local varieties of mulberry based on the genetic similarity using ISSR markers was generated by UPGMA cluster method. In the dendrogram, most varieties from the same ecotype clustered together.