Analysis of genetic diversity in Portuguese Ceratonia siliqua L. cultivars using RAPD and AFLP markers

Although carob (Ceratonia siliqua L.) is of great economic importance little is still known about the pattern of genetic variation within this species. Morphological characteristics based on 31 fruit and seeds of continuous characters determinant for agro-industrial uses, were compared with RAPD and AFLP markers for assessing genetic distances in 68 accessions of carob trees, from different cultivars, varieties and eco-geographic regions of Algarve. Eighteen selected RAPD primers applied to the 68 accessions produced a total of 235 fragments ranging from 200 to 2000 bp, of which 93 (40%) were polymorphic. Four AFLP selective primer combinations generated a total of 346 amplification fragments of which 110 were polymorphic. The average level of polymorphism based on four primer combinations was 31.8%. The phenetic trees based on RAPD and AFLP analyses gave high co-phenetic correlation values, and were found to be consistent in general with the analysis of morphological data, carried out on the same accessions. A number of RAPD and AFLP markers were found to be diagnostic for ‘Canela’ cultivar and 13 wild ungrafted trees.     

Evaluation of fruit and seed diversity and characterization of carob (Ceratonia siliqua L.) cultivars in Algarve region

The genetic diversity of 15 carob (Ceratonia siliqua L.) cultivars located in an experimental field from Algarve (Portugal) was evaluated over 7 years using 12 fruit and seed phenotypic characters, in order to characterize carob cultivars. The values of morphological traits obtained by cultivar were compared with those from other countries of the Mediterranean basin. Statistically significant differences were found between cultivars for all characters which were examined, what indicates a high genetic diversity. The relationship among these characters was analyzed by principal component analysis (PCA) resulting in the separation of these cultivars classed in four groups (clusters I–IV) and in four ungrouped cultivars. A three dimension of the model was found to be significant and explained 74.5% of the total variation, in which the first component accounting for 34.6% of the total variation is dominated by fruit characters, while the second component is dominated by seed characters. Cultivars plotted on the left-lower quadrant on the space determined by principal components 1 and 2 are characterized by fruits with high seed yield more appropriated for industrial rentability. The correlation analyses established by cultivar provided a specific understanding about the way how fruit and seed characteristics correlate within each cultivar. This approach can be useful for the development of a breeding programme, aiming to increase the seed yield, seed thickness, individual and total seed weight by fruit, characteristics that are determinant to improve the industrial exploitation of carob.     

Fruit weight is related to ovary weight in olive (Olea europaea L.)

Fruit size is an important parameter both for scientific understanding and for commercial purposes. In many species, mature fruit size is often related to floral ovary size, but no literature exists in olive that demonstrates such a relationship. Previous work suggests that olive cultivars with different fruit sizes have similar cell number and size in the ovary transectional area, but ovary and fruit dry weight was not measured. In the present study, ovary dry weight and fruit dry weight during the whole fruit development season until harvest were measured in olive cultivars with different fruit size, over three years. Flower dry weight was also measured. Fruit weight at harvest was strongly correlated to ovary weight at bloom, both in single-year data and when data from three years were pooled. Flower dry weight, excluding the ovary, was also correlated to ovary dry weight. Ovary dry weight was strongly correlated not only to the fruit dry weight at maturity, but also at any date during fruit development. The mature fruit/ovary dry weight ratio ranged between 1000 and 4000 among cultivars, but was not correlated to the fruit dry weight at maturity. These results suggest that, in olive, fruit weight is genetically controlled through the ovary weight at bloom. This knowledge may have implications in the understanding of fruit set and source-sink relationships in olive.     

Lipid evaluation of cultivated and wild carob (Ceratonia siliqua L.) seed oil growing in Turkey

Studies were carried out on the oil content, fatty acids, tocopherols and sterols in the seeds of two carobs (Ceratonia siliqua), cultivated and wild, from Turkey. The oil yields of cultivated and wild carob seeds were established as 1.73 and 1.82% on a dry weight basis, respectively. The main fatty acids in cultivated and wild carob seed oils are linoleic (49.1% and 51.0%), oleic (30.4% and 26.5%), palmitic (10.3% and 12.0%) and stearic (3.5% and 4.6%), respectively. Tocopherols and phytosterols are important constituents of the unsaponifiable fraction of several seed oils. The major tocopherol in both seed oils was γ-tocopherol. The composition (mg/100 g) was as follows α-tocopherol (69.06 and 70.39 mg/100 g), P8 (22.29 and 24.78 mg/100 g), δ-tocopherol (8.70 and 10.66 mg/100 g), β-tocopherol (2.30 and 1.85 mg/100 g). The total tocopherol contents had 208.45 and 223.14 mg/100 g, respectively. The total content of sterols of both oils were determined as 16400.94 and 30191.55 mg/kg, with β-sitosterol as the predominant sterol that accounted for more than 70% of the total amount of sterols other sterols, campesterol (5.33–5.32%), stigmasterol (0.58–11.43%) 7-avenasterol (3.45–3.03%), 7-stigmasterol (2.16–2.4%), and chlerosterol (1.33–1.0%) were detected in both carob oils. As a result, the accurate quantification of these analyses has very important applications for the nutrition sciences.     

Genetic analysis of Tunisian fig (Ficus carica L.) cultivars using amplified fragment length polymorphism (AFLP) markers

Genetic diversity of forty fig cultivars collected from five regions in Tunisia was investigated using amplified fragment length polymorphism (AFLP). A total of 342 reproducible bands amplified with six AFLP primer combinations were obtained. The high percentage of polymorphic bands (%PB) of 97.5 and the resolving power (Rp) collective rate value of 143 were scored. In addition, the polymorphism information content (PIC) values varied from 0.61 to 0.87 with an average of 0.77. Although cluster (UPGMA) and principal components analyses indicate that the cultivars’ clustering made independently both from the geographical origin, horticultural classifications and/or from the sex of trees. In addition, the observed variation suggests considerable differentiation among fig cultivars. The present data supports the common origin of the fig cultivars. Analysis of molecular variance (AMOVA) revealed that average Φ_ST value overall loci was 0.026, and the overall distribution pattern of molecular variation indicated that about 97.43% of the total variance was accounted by the within-region variance component. The remaining 2.5% (P < 0.001) of the variation was founded among cultivars of the prospected regions. Our results proved that AFLP markers are useful for germplasm discrimination as well as for investigation of fig patterns variation. The information may be useful to define conservation management program.     

Direct and indirect effects of seed related characters on number of seed in guava (Psidium guajava L.) fruits

Dessert quality of guava fruit is considerably reduced by high seed content. Number of seeds in guava is associated with different seed and fruit characters. Influence of different seed characters on number of seeds per fruit (NSPF) was studied by analysing character association and direct and indirect effects on NSPF and seed related traits for 68 genotypes of guava collected from diverse sources and conserved at National Active Germplasm Site, CISH, Lucknow. Seed related characters were studied at the colour break stage of the fruits using image analysis software for counting number of seeds. The data was subjected to path analysis to find out direct and indirect effects of different characters on number of seeds in the fruits. At genotypic and phenotypic levels, NSPF was significantly and positively associated with seed weight per fruit (SWPF), number of seeds 100 g⁻¹ pulp (NSPHP) and fruit weight (FW). The genotypic (0.0029) and phenotypic (0.0563) residual values were fairly very low, which revealed that variables included in this study had significant contribution in determining NSPF. The NSPHP exhibited high positive direct effect on NSPF. The maximum direct response (P = 0.737, G = 1.004) of this component was attributed to the indirect positive effects of the SWPF and fruit:seed weight proportion (FSWP). SWPF also had a very high positive direct effect (P = 0.521, G = 0.694) on the NSPF. Correlation and path coefficient analysis revealed that SWPF, NSPHP and 100-seed weight (100SW) were deciding factors for realizing improvement for NSPF. The importance of small seeded genotypes in selection of less seeded varieties was emphasized.     

Molecular based assessment of genetic diversity within Barbary fig (Opuntia ficus indica (L.) Mill.) in Tunisia

In this work, we report for the first time on the analysis of genetic diversity within a set of 36 Tunisian Opuntia ficus indica (L.) Mill. ecotypes using RAPD markers.     

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.     

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 diversity in local Tunisian pears (Pyrus communis L.) studied with SSR markers

Few records are available about local Tunisian pear cultivars characterized by low chilling requirements and adaptation to dry conditions. In this work, seven SSRs derived from apple were successfully transferred to 25 local Tunisian pear genotypes and 6 common varieties of Pyrus communis cultivated in Europe. The 7 SSRs used amplified a total of 36 fragments. All the microsatellites except one seem to amplify more than one locus in some of the genotypes studied. Only 12 different fingerprinting patterns could be distinguished among the 25 Tunisian cultivars studied indicating a high number of synonymies. The mean expected and observed heterozygosities in the 25 Tunisian cultivars analyzed averaged 0.71 indicating a high level of genetic diversity among the local Tunisian pear germplasm. These markers will be useful to optimize the conservation of this highly threatened germplasm.     

Isolation and characteristics of eight novel polymorphic microsatellite loci from the genome of garlic (Allium sativum L.)

Here we characterized eight novel polymorphic SSR markers, developed from an enriched genomic library of garlic (Allium sativum L.). These SSRs produced a total of 64 alleles across 90 garlic accessions, with an average of 8 alleles per locus. Values for observed (H_O) and expected (H_E) heterozygosity ranged from 0.16 to 0.77 (mean = 0.44) and from 0.22 to 0.86 (mean = 0.65), respectively. Six loci deviated significantly (P < 0.05) from Hardy–Weinberg equilibrium (HWE). The averages of gene diversity and PIC values were 0.65 and 0.62, respectively. The mean genetic similarity coefficient was 0.4380, indicating that among garlic accessions existed wide genetic variation. Based on 64 alleles obtained by 8 SSRs, a phenogram was constructed to understand the relationships among the 90 accessions. These newly developed SSRs should prove very useful tools for genotypes identification, assessment of genetic diversity and population structure in garlic.     

Biocontrol of mildew with Bacillus subtilis in bitter gourd (Momordica charantia L.) seeds during germination

The bitter gourd seed has a thick, hard seed coat. Mildew often occurs during germination and causes uneven and low rates of seed germination. However, the problems caused by mildew can be overcome by treating seeds with hot water, by soaking in water, or by using microorganisms. Seeds of the ‘Ching Pi’ bitter gourd were treated in a water bath at 60 °C for 10 min and then soaked in tap water at 25 °C for 24 h. The resulting germination percentage was 86.7%, and the resulting percentage of mildewed seeds was 10%. The biocontrol potential of three commercially available Bacillus subtilis solutions was examined. For seeds primed with Huodijun B. subtilis solution, the germination percentage was 73.3% and the mildewed percentage 6%. In dual cultures, the antibiotic content in the Huodijun B. subtilis solution was significantly greater than in Yunghsing and Huolibao, the other B. subtilis solutions examined. B. subtilis effectively inhibited the growth of Rhizoctonia solani mycelium and caused abnormal mycelial growth.     

In vitro embryo germination and proliferation of pistachio (Pistacia vera L.)

In vitro development of isolated embryos and axillary bud proliferation were studied in Pistacia vera L. Different regulator-free nutrient media were compared to determine the effects of the mineral solution, agar and sucrose concentrations on seedling development from mature embryos. Nutrient-rich MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tabacco tissue cultures. Physiol. Plant. 15, 473–479] and DKW [Driver, J.A., Kuniyuki, A.M., 1984. In vitro propagation of Paradox walnut rootstock. HortScience 19, 507–509] mineral solutions were more efficient for the development of aerial parts than nutrient-poor KN [Knop, W., 1884. Bereitung einer concentrierten nährstofflosung für pflanzen. Landwersuhssat 30, 292–294] and WT [Withe, P.R., 1936. Plant tissue cultures. Bot. Rev. 2, 419–437] solutions. Reducing the agar concentration enhanced fresh matter production and balanced seedling development. When tested at different concentrations, sucrose was found to orient mature embryo development, with the best results obtained at concentrations of 2–4%, whereas high concentrations (6 and 12%) mainly inhibited elongation of the aerial parts. Plantlets obtained previously from in vitro cultured embryos were propagated by axillary budding. High bud proliferation (six shoots per explant) was achieved when using 17.8 μM benzyladenine (BA) combined with 0.5 μM indole-3-butyric acid (IBA). The addition of 2.9 μM gibberellic acid (GA₃) to the propagation medium did not improve axillary shoot yields and on average, media with GA₃ produced 2.3–2.6 elongated stems per cultured explant. Shoots were rooted in vitro in half-strength MS medium containing 12.3 μM IBA.     

Assessing Rosa persica genetic diversity using amplified fragment length polymorphisms analysis

The genetic diversity among 128 Iranian Rosa persica (R. persica) accessions in the different populations was analyzed. Amplified fragment length polymorphisms (AFLP) technique was used to produce 171 polymorphic fragments. The number of polymorphic loci ranged from 101 to 147 and the polymorphism information content (PIC) varied from 0.289 to 0.073, with an average of 0.16. This shows extreme variability and genetic diversity among the studied R. persica populations. An indirect estimate of the number of migrants per generation (Nm = 0.376) indicated that gene flow was relatively low among populations of the species. Cluster analysis using the UPGMA method grouped all accessions into six clusters. The results did not show relative agreement with the genotypes’ region of origin. Based on an analysis of molecular variance, 48% of the genetic variation of R. persica was within population and 52% was among populations. The present analysis revealed that Iranian R. persica genotypes are highly variable and genetically distinct from their origins. The apparent unique nature of the R. persica genotypes revealed by our results supports the case for the implementation of more intense characterization and conservation strategies, and provides useful information to address breeding programmes and germplasm resource management in Rosa spp.     

Effects of growing region and maturity stages on oil yield and fatty acid composition of coriander (Coriandrum sativum L.) fruit

Changes on oil yield and fatty acid profiles were studied during maturation of coriander (Coriandrum sativum L.) fruits cultivated in Menzel Temime and Oued Beja, Tunisia. Oil and petroselinic acid biosynthesis proceeded at a steady rate up to 16 DAF in Oued Beja and in 33 DAF in Menzel Temime. The first results show that a rapid oil accumulation started at newly formed fruits and continued until their full maturity. During fruit maturation, fatty acid profiles varied significantly among the growing regions and stages of maturity. Petroselinic acid had the highest amount at the 16th and the 33th DAF, in Oued Beja and Menzel Temime, respectively. In Oued Beja, at full maturity, the main fatty acids were petroselinic acid (80.90 ± 9.45%), followed by oleic (14.79 ± 2.25%), palmitic (3.50 ± 0.65%) and stearic (0.49 ± 0.09%) acids. Fatty acid profile of fruits cultivated in Menzel Temime showed that in fully ripe fruit, petroselinic acid is the main compound (80.86 ± 7.23%) followed by oleic (14.83 ± 2.05%), palmitic (3.27 ± 3.12%) and stearic (0.31 ± 0.05%) acids. In both growing region, fruit development resulted mainly in an increase of petroselinic acid and a decrease of palmitic acid. Saturated and polyunsaturated fatty acids decreased significantly and monounsaturated fatty acids increased during maturation of fruit. Oil composition at the first four stages of maturity has a healthy and nutritionally value and the last stages were with important economic and industrial applications. Coriander fruit is potentially an important source of petroselinic which have numerous industrial applications.     

Callus induction and plant regeneration from cotyledonary explants of ash gourd (Benincasa hispida L.)

A protocol for the production of complete plantlets through multiple shoots from the cotyledon-derived calli of ash gourd (Benincasa hispida L.) is described. The embryos were excised from mature seeds and cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurin (BAP, 1–5 μM). After 10 days the well-developed green cotyledons from the growing embryos were isolated and cultured on MS medium fortified with 2,4-D (1–6 μM). The cultured cotyledons gave rise to luxuriantly growing calli after 6 weeks. These calli were subcultured on MS medium supplemented with various concentrations of BAP (1–6 μM) alone or in combination with naphthalene acetic acid (NAA, 0.2 and 0.5 μM) for regeneration. The regenerated shoots were multiplied and rooted on quarter strength MS medium supplemented with indole-3-butyric acid or NAA (1–5 μM). The rooted shoots were transplanted to soil with 90% success.     

Fatty acids, bioactive lipids and radical scavenging activity of Celastrus paniculatus Willd. seed oil

In the present contribution lipid composition and characteristics of Indian Celastrus paniculatus seed oil were determined. C. paniculatus seeds are a rich source of oil (46%). The amounts of neutral lipids in the oil were the highest, followed by glycolipids and phospholipids. Oleic followed by palmitic and linoleic were the major fatty acids in C. paniculatus seed oil and its lipid classes. C. paniculatus seed oil being characterized by a relatively high amount of phytosterols, wherein the sterol marker was β-sitosterol followed by campesterol and stigmasterol. γ-Tocopherol was the major tocopherol isomer while the rest being α-tocopherol. When C. paniculatus oil and extra virgin olive oil were compared upon their radical scavenging activity (RSA) toward the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical C. paniculatus oil exhibited stronger RSA. In consideration of potential utilization, detailed knowledge on the composition of C. paniculatus oil is of major importance. The diversity of applications to which C. paniculatus oil can be put gives this plant great industrial importance.