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.     

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.     

Antioxidant response of bitter gourd (Momordica charantia L.) seedlings to interactive effect of dimethoate and UV-B irradiation

Bitter gourd (Momordica charantia L.) seedlings treated with elevated concentrations of dimethoate (100 and 200 ppm) and fixed ultraviolet-B (0.4 Wm⁻²/30 min) irradiation showed stunted growth and less photosynthetic pigments chlorophylls (Chl) content. The synergistic effects of both the stresses were more pronounced than the individual effect. However, dimethoate at low concentration (50 ppm) stimulated growth and pigmentation but with UV-B it showed slight inhibition. Reactive oxygen species (ROS) accumulated considerably in leaves due to UV-B and high concentrations of dimethoate. Combined exposure further increased the ROS leading to lipid peroxidation and electrolyte leakage. Both the stresses alone and together also caused the increase activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). High concentration of dimethoate and UV-B accelerated the accumulation of ROS particularly H₂O₂ in leaves, causing heavy damage to photosynthetic pigments and growth of bitter gourd seedlings. Simultaneous exposure of UV-B and dimethoate inhibit the growth, photosynthetic pigment and enhanced the accumulation of ROS more severely than the individual exposure. Interestingly, low concentration (50 ppm) of dimethoate significantly reduced the effects of UV-B. The results suggested synergistic effect of dimethoate and UV-B on plant growth as a function of decreased photosynthetic pigments despite increase in the activities of the antioxidant enzyme.     

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.     

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.     

A comparative analysis of the genetic diversity between inbred lines of Zinnia elegans using morphological traits and RAPD and ISSR markers

Genetic diversity within Zinnia elegans is key to the genetic improvement of this important ornamental species. Here, morphological traits and RAPD and ISSR molecular markers were used to assess levels of polymorphism across 20 inbred lines. Thirty-four morphological traits were scored and also 147 RAPD marker-fragments, as amplified by 12 arbitrary primers, and 128 ISSR marker-fragments as generated by 9 primers. The number of polymorphic loci, the percentage of polymorphic loci, Shannon's Information index (I) and the effective number of alleles (Ne) were calculated from the RAPD data as 100, 68.03%, 0.3559 and 1.4169, respectively. From the ISSR data, these respective statistics were calculated as 97, 76.38%, 0.4013 and 1.4728. Thus, ISSR markers were considered slightly superior to RAPD markers for assessing genetic diversity between the accessions; however, Mantel's test indicated significant correlation (R = 0.733) of the RAPD and ISSR results. By contrast, the morphological matrix showed low correlation with both RAPD and ISSR data matrices (R = 0.3814 and 0.3765, respectively). Cluster analysis showed that groupings of the accessions according to all three methods correlated well with their geographic region of origin, but flower color was not strongly associated with the genetic classification of these inbred lines of Z. elegans.     

Comparative analysis of genetic diversity in Prunus L. as revealed by RAPD and SSR markers

RAPD and SSR markers were used for genetic diversity evaluations among 15 genotypes selected from the genus Prunus L. Altogether 40 RAPD primers and 21 primer pairs designated for microsatellite loci were applied on the whole group of genotypes.     

Assessment of genetic diversity in cashew germplasm using RAPD and ISSR markers

Diversity and genetic relationship in 100 cashew germplasm accessions were analyzed by using RAPD and ISSR markers. Using 10 selected RAPD primers 60 bands were generated, of which 51 bands were polymorphic (85%), and with 10 selected ISSR primers 67 amplified bands were observed with 58 polymorphic bands (86.6%). Though both kinds of markers discriminated the accessions effectively, analysis of combined data of markers (RAPD + ISSR) resulted in better distinction of accessions. By combining markers, a total of 127 bands were detected, of which 109 bands (85.8%) were polymorphic and produced on an average of 5.45 polymorphic bands per primer. Primers with high polymorphic information content and marker index were identified for discriminating accessions. High percentage of polymorphism (>85%) observed with different markers indicated high level of genetic variation existing among the accessions. Genetic relationship estimated using similarity co-efficient (Jaccard’s) values between different pair of accessions varied from 0.43 to 0.94 in RAPD, 0.38 to 0.89 in ISSR and 0.43 to 0.87 with combined markers suggested a diversity (dissimilarity) ranging from 6 to 57%, 11 to 62% and 13 to 57% respectively and the diversity skewed around 50% indicated moderate diversity. The cluster analysis with UPGMA method separated the accessions broadly into 13 clusters and in that three into smaller clusters. Some correspondence between the molecular groupings and the morphological clusters were observed. Among the accessions, NRC-142 and NRC-12 were highly divergent and NRC-231 and NRC-232 were genetically similar.     

Genetic diversity in Indian ash gourd (Benincasa hispida) accessions as revealed by quantitative traits and RAPD markers

In present study, an attempt has been made to assess the genetic diversity among 34 accessions of Benincasa hispida using quantitative traits and RAPD data. Variability was observed for characters like days to first female flower, fruit weight, equatorial and polar diameter. Cluster analysis based on quantitative traits revealed a high degree of diversity among the accessions. RAPD-based dendrogram showed dissimilarity values 0.64–0.943 suggesting that the accessions represent a genetically diverse population. A non-significant correlation was observed between the clustering based on quantitative traits and RAPD markers. But the accessions like IVAG-107 and IVAG-81 clearly separated from rest of the accessions based on the quantitative traits and as well as RAPD primers. Both the dendrogram revealed that the accessions from northeastern region of India are diverse from the accessions of other parts of India, as they cluster together in both the dendrogram. The information generated could be utilized in developing cultivars/hybrids for specific traits.     

Microsatellite markers based assessment of genetic diversity in Iranian olive (Olea europaea L.) collections

To study the genetic variation in Iranian olive collections and some foreign olive cultivars, 47 accessions of 18 local cultivars from 6 olive collections of Iran (Roudbar, Zanjan, Ahvaz, Dezful, Kazeroon and Shiraz), were analyzed along with 30 imported cultivars using 16 microsatellite primer pairs. All the used microsatellite loci revealed polymorphism in the studied genotypes, except GAPU14 and GAPU113 markers. Fourteen microsatellite primers amplified 126 polymorphic alleles in the 87 selected olive accessions. The average number of alleles per locus was 9, ranging from 3 to 14. Polymorphic information content (PIC) was 0.85. The genetic similarity based on Jaccard coefficient ranged from 0.15 to 1. The genetic relationships among accessions were investigated using cluster analysis and principal component analysis (PCA). Most of the accessions with the same name were grouped together; some exceptions were also observed. As expected, close relationship was observed among accessions within same cultivar. Most of the Iranian olive accessions were clustered to a main distinct group. Two-dimensional scatter plot of principal component analysis revealed a clear separation of most of the Iranian olives from Syrian and other introduced cultivars. These suggest that Iranian cultivars have different origin related to West Mediterranean basin cultivars and have evolved independently from the others. Between and within Iranian and foreign cultivars (cultivars including three or more accessions) genetic diversity was analyzed using analysis of molecular variance (AMOVA). AMOVA revealed higher within cultivar genetic variation (62.76%) as compare to that between cultivar variations (37.24%). The intra- and inter-cultivar variance tested by permutation test showed significant genetic variation at both levels. The high level within cultivar genetic variance could be due to mislabeling and presence of homonyms in cultivars produced by vegetative propagation from original plants.     

Genetic diversity of Chinese natural bermudagrass (Cynodon dactylon) germplasm using ISSR markers

Bermudagrass (Cynodon dactylon L.) is an important warm-season turfgrass. Although it is widely distributed in China, studies on the genetic variation and relationship among the large-scale indigenous bermudagrass were relatively insufficient, especially at molecular level. The purpose of this study was to assess the molecular variation and relationship among one commercial cultivar ‘Tift3’ and 95 wild bermudagrass accessions collected from 11 provinces in China by ISSR marker technique. The results indicated that 29 ISSR primers generated a total of 248 bands among which 242 (97.6%) were polymorphic bands. The genetic similarity coefficients among accessions ranged from 0.51 to 0.97 with an average of 0.74. All accessions could be clustered into 11 groups with UPGMA method. Accessions from the same or adjacent regions generally were clustered into the same group or subgroups. A few accessions, however, were greatly different from the majority of all accessions. The results suggested that ISSR marker is an effective tool for the study of genetic variation in Chinese natural bermudagrass.     

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.     

Genetic diversity and population genetic structure of pomegranate (Punica granatum L.) in Iran using AFLP markers

Pomegranate (Punica granatum L.) is one of the oldest known edible fruits. It is native to Iran and spread from Iran to other areas. In this study amplified fragment length polymorphism (AFLP) was used to detect intra- and inter-population genetic diversity of pomegranate. A group of 67 accessions belonged to 4 populations from Iran was studied using eight primer combinations. A total of 221 scorable bands were amplified, of which, 118 (54.13%) were polymorphic. Resolving power (Rp) ranged from 5.70 to 9.21, and the average of polymorphism information content (PIC) per primer pair was 0.40. According to Nei's gene diversity and allelic statistics, Isfahan population had a highest genetic diversity (H = 0.3646, I = 0.5327, N_e = 1.6467). Coefficient of gene differentiation between populations (GST) was 0.124, indicated that mainly proportion of genetic variation (87.6%), was within populations and the remaining (12.4%) of the variation was among populations that, also supported by analysis of molecular variance (AMOVA). The gene flow (Nm) varied from 0.969 to 10.404 between pair-wise populations and was 3.504 among all of the populations. The Jaccard similarity coefficient between individuals ranged from 0.26 to 0.88. The UPGMA dendrogram clustered all 67 accessions into 6 groups. In some cases accessions from same region were grouped together but in most cases, there was gene exchange. To study the genetic relationships among populations, a principal coordinate analysis (PCoA) based on Nei's genetic distances was performed. Results of this study showed that AFLP marker can be a useful tool for investigating the genetic diversity of pomegranate genotypes.     

RAPD analysis of genetic diversity among and within Portuguese landraces of common white bean (Phaseolus vulgaris L.)

Seventeen landraces of common beans (Phaseolus vulgaris L.) sampled in the North and Centre of Portugal were analyzed at population level for 689 RAPD loci amplified by using forty random primers. The two different parameters used to estimate the genetic variability in and between samples indicate that the inter-population component of the genetic variability is mainly responsible for the diversity found, since only about a 10% would be of an intra-population nature. In addition, the gametic disequilibrium was estimated and reached an average value of 40% for the different combinations of pairs in the 689 loci studied taking the 17 samples as a whole population. Self-pollination, genetic drift and adaptation would thus be favouring the formation of multilocus associations. In addition, the fingerprinting study suggests that each landrace produced unique amplification products allowing it to be distinguished from the other tested genotypes, but, nevertheless, the landraces show a considerable genetic similarity (56% and 70.5% using two different methods). The Neighbour-Joining dendrogram did not show any relation between the geographical distribution of landraces and genetic distance. The results suggest that these Portuguese landraces conserved an important genetic diversity that can be useful to widen the genetic base of currently cultivated beans.     

Identification and genetic relationship based on ISSR analysis in a germplasm collection of sea buckthorn (Hippophae L.) from China and other countries

Sea buckthorn (Hippophae L.) is a woody, outcrossing dioecious pioneer plant, being widely planted as a new berry crop with rich nutritional and medicinal compounds, as well as a means of prevention of soil erosion. Inter simple sequence repeat (ISSR) markers were generated in 219 plants of 40 cultivars from China and other countries, representing two species of Hippophae salicifolia and Hippophae rhamnoides that includes two subspecies (ssp. mongolica and ssp. rhamnoides) and intraspecific hybrids between ssp. mongolica and ssp. sinensis. Fifteen selected ISSR primers generated 385 bands with an average of 25.6 bands per primer. Thirty hundred and eighty-five bands were used to generate a Dice's coefficient matrix of pairwise comparisons between individual ISSR profiles. Cluster analysis based on this matrix showed clustering of plants into groups which was in good agreement with their taxonomic classification. The results also revealed the presence of different cultivars with the same name, identical cultivars but with a different name and the genetic relationship of cultivars with unknown parentage to those with known parentage. Our data will be helpful in collection and conservation of sea buckthorn germplasm and potential hybrid strategy for breeding.     

Application of RAPD and SI-typing techniques to confirm genetic integrity of radish (Raphanus sativus L.) varieties

The random amplified polymorphic DNA (RAPD) and self-incompatibility (SI) typing techniques were applied to settle a lawsuit between a Seed Company and a farmer concerning seed impurity in radish (Raphanus sativus L.). After sowing “Halra Woldong” (HW) a winter radish variety, two morphologically different radish plants grew. Genomic DNA from each of the two types were prepared and analyzed using ninety-six RAPD markers and four variety-specific markers were found. The polymorphic RAPD band patterns were compared with those of the twenty Korean radish varieties. The genetic integrity of the two varieties in question was identified and further confirmed by SI-typing of S-locus glycoprotein (SLG) and S-locus receptor kinase (SRK) genes.     

Genetic diversity analysis in date palm (Phoenix dactylifera L.): a comparative assessment using ISSR and RAPD marker assays

SUMMARY

A comparative study was conducted to evaluate genetic diversity in 45 genotypes of date palm (Phoenix dactylifera L.), including both male and female plants, employing RAPD and ISSR marker systems. The data were analysed to calculate the total number of bands, the number of polymorphic bands, the percentage polymorphism, the average number of bands per primer, the effective multiplex ratio (EMR), the polymorphic information content (PIC), the marker index (MI), and genetic similarity coefficients. The 37 RAPD and 53 ISSR primers used generated 363 and 608 scorable amplified products, respectively, of which 95.0% and 90.9% were polymorphic. The ISSR markers produced more information than the RAPD markers due to their higher EMR and MI values. Jaccard similarity values among male plants, female plants, and between all male and all female plants varied between 0.72 – 0.80. The results indicate the effectiveness of these two marker systems for demonstrating genetic relationships among date palm genotypes.     

Utility of RAPD,ISSR, IRAP and REMAP markers for the genetic analysis of Citrus spp.

The application and informativeness of RAPD, ISSR, IRAP and REMAP markers to study the genetic diversity and relationship among the Citrus and its relative genotypes were investigated. High levels of polymorphism were observed for all four marker systems. The RAPD technique generated the highest number of polymorphic bands and average number of polymorphic band per assay unit. Average limit of the discriminating power was very close to its actual discriminating power of each marker. The highest and the lowest values of effective number of patterns were obtained from the marker REMAP (5.94) and RAPD (4.48), respectively. Correlation between the genetic similarities matrices were estimated from all four markers using the Mantel matrix correspondence test, and results showed significant correlations among the RAPD, ISSR, IRAP and REMAP similarity matrices. The highest correlations were found comparing RAPD and ISSR markers, whereas RAPD and REMAP (r = 0.143) markers were poorly correlated. To assess the usefulness of the overall information provided by these marker data for establishing phylogenetic relationships and Citrus germplasm classification, cluster analysis was performed. All four techniques, solely or in combination, discriminated the genotypes very efficiently and generated a high similarity in dendrogram topologies, although some differences were observed. The linkage analysis was conducted based on the segregation of 38 RAPD, 13 ISSR, 19 IRAP and 9 REMAP loci in 96 progeny of an intergeneric cross Citrus sinensis and Poncirus trifoliata. Among the 81 studied loci 65 loci distributed on five linkage groups. Comparing the result obtained with RAPD, ISSR, IRAP and REMAP markers in this study, IRAP and REMAP proved to be as a reliable molecular marker for fingerprinting, mapping and diversity study of Citrus and its relatives.     

Microsatellite characterization of grapevine (Vitis vinifera L.) genetic diversity in Asturias (Northern Spain)

The genetic heritage of the Asturian grapevine (Vitis vinifera L.) has been declining over the past century due to the phylloxera attack and the further abandonment of this culture. In addition, efforts in recent years to restore the Asturian vineyard with the pulling-up of old vineyards and replanting with cultivars endorsed by Cangas Quality Wine regulations are contributing even more to this genetic erosion. The aim of this study was the evaluation and identification of the phytogenetic resources of the Asturian grapevine. A total of 293 accessions were collected in old vineyards and analyzed through nine microsatellite markers. Forty-two different genotypes were obtained, including six profiles with allelic variations. Only 27 cultivars were identified when compared with national and international databases; some of them had not been found in this region before. Homonymies and synonymies have also been detected. These results provide an overview of the status of current grapevine phytogenetic resources in Asturias. Despite the substantial genetic erosion that the Asturian vineyard has suffered, a higher variability than expected has been detected. The finding of new grapevine genotypes is a fact of great importance. The genetic grapevine resources are being drastically reduced all over the world, so this new genetic material has to be included in germplasm banks for its conservation and further agronomical and enological evaluation.     

Genetic diversity evaluation of wild Persian shallot (Allium hirtifolium Boiss.) using morphological and RAPD markers

Persian shallot, a bulb producing plant from Alliaceae, is a wildly growing plant collected for its bulbs. Bulbs of Persian shallot, called “Mooseer” in Farsi, are oval, white skinned, usually of one and rarely of two main bulbs and are completely different from common shallot (Allium ascalonicum). There is no information about genetic diversity of this species; therefore, the diversity of 17 wild Persian shallot populations collected from different parts of Iran across the Zagross Mountains were evaluated by morphological and RAPD markers. Fifteen random decamer primers could amplify DNA well and produced polymorphic bands among populations. The most fragment number amplified by one primer was 27 out of which 15 fragments were polymorphic and the least was 11 with 6 polymorphic bands. At the similarity of 0.60 on dendrogram constructed on the base of RAPD data, the samples were divided into eight sub-clusters. The highest and lowest detected similarities were 0.73 and 0.49, respectively. Clustering based on morphological traits divided the populations into three groups. Result of cluster analysis based on RAPD data did not show any correlation with morphological characters based on Mantel’s test (r = 0.03). The mineral and fatty acid analysis for defining the nutrient composition of this plant also revealed some differences among populations.