Analysis of genetic diversity among Indian short-day onion (Allium cepa L.) cultivars using RAPD markers.

Summary

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate genetic diversity among 24 cultivars of short-day onions. Total genomic DNA was extracted and subjected to RAPD analysis using 90 arbitrary 10-mer primers. Of these, 15 primers were selected which yielded 137 bands, 91.24% of which were polymorphic. None of the primers produced a unique banding pattern for each cultivar. RAPD data were used to calculate a Squared-Euclidian Distance matrix which revealed a minimum genetic distance between cultivars ‘AFLR-722’ and ‘PBR-140’, and a maximum genetic distance between cultivars ‘PBR-139’ and ‘A.Kalyan’, and ‘MS-48’ and ‘A.Kalyan’. Based on the distance matrix, cluster analysis was done using a minimum variance algorithm.The dendrogram thus generated, based on Ward’s method, grouped the 24 onion cultivars into two major clusters. The first cluster consisted of cultivars from the northern region, and the second of cultivars from the southern region of India. The present study shows that there is high diversity among the onion cultivars selected and indicates the potential of RAPD markers for identification and maintenance of onion germplasm for crop improvement purposes.     

Inter- and intra-varietal analysis of three Olea europaea L. cultivars using the RAPD technique

Summary

Characterization and selection of olive clones for the production of olive oil is essential in Portugal because of its profitable exploitation. “Moura-Serpa” is the most important Portuguese region for the production of olive oil relying on three cultivars for oil quality. These are ‘Galega Vulgar’, ‘Cordovil de Serpa’ and ‘Verdeal Alentejana’. Therefore, it is of great importance to guarantee the varietal certification of the young trees and the establishment of new orchards. Random Amplified Polymorphic DNA (RAPD) technique was used to characterize these three cultivars. Analysis started using twenty primers that allowed us to distinguish the three cultivars and to select a reduced set of primers. The selected primers were used for inter- and intra-varietal analysis and for establishing a profiling system to assay genetic diversity in other olive cultivars. The method has the potential for use in varietal certification and breeding programmes that need to analyse a high number of samples.     

A PCR-based assessment of genetic diversity,and parentage analysis among commercial mango cultivars and hybrids

Summary

Three different PCR methods [Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeats (ISSR), and Directed Amplification of Minisatellite DNA (DAMD)] were used to analyse genetic diversity and parentage among 20 mango cultivars, including 18 landraces and two hybrids (‘Amrapali’ and ‘Mallika’). These hybrids together with a third hybrid (‘Ratna’), and an out-group species (Mangifera sylvatica) were also analysed for parentage. Fifteen, seven and four primers were used to amplify a total of 158, 69 and 59 distinct DNA fragments by RAPD, ISSR and DAMD, respectively. Of these, approx. 85%, 64% and 90% were polymorphic, respectively. Genetic distances between pairs of mango cultivars were measured separately by each method and depicted graphically as a Neighbor Joining (NJ) tree. The three methods revealed different groupings of cultivars and hybrids. A NJ tree based on the cumulative data from all methods correlated well with the parentage of the mango hybrids, and the grouping of cultivars on a regional basis. Genetic markers likely to be associated with important agronomic traits were identified by further analysing the hybrids, with their respective parents, using all three methods. On the basis of the highest number of polymorphic bands observed (90%), DAMD was judged to be the best method with which to analyse mango germplasm.     

Morphological and molecular analysis of genetic diversity in jackfruit

Summary

RAPD markers were used to estimate genetic diversity in 12 high-yielding jackfruit (Artocarpus heterophyllus Lamk.) accessions obtained from different locations in southern India. Marker data were compared with morphological data obtained over three successive seasons. PCR-amplifiable DNA was isolated using the CTAB method and 171 amplified fragments were obtained using 23 random primers. The genetic dissimilarity matrix was calculated based on Squared Euclidian Distances, which revealed a maximum genetic distance of 7.9% between a clone of ‘Mottavarica’ (‘M₀’), and ‘Chandrahalasu’ from distant locations, while the minimum genetic distance (5%) was between the genotypes (‘M₀’) and ‘Kerala’, indicating their similar geographical origin. Ward's method of cluster analysis grouped all individuals on the dendrogram into two major clusters according to their geographical location. The present study showed low-to-moderate genetic diversity among the 12 jackfruit accessions, which will assist in the identification and management of jackfruit germplasm for breeding purposes.     

Detached Cane Assay of Resistance to Botryosphaeria Cane Canker (Botryosphaeria dothidea) in Eastern U.S. Blackberry Genotypes

Abstract

Resistance to Botryosphaeria cane canker in blackberry (Rubus subgenus Rubus Watson) was studied in eleven cultivars (‘Apache’, ‘Arapaho’, ‘Chester Thornless’, ‘Chickasaw’, ‘Illini Hardy’, ‘Kiowa’, ‘Navaho’, ‘Ouachita’, ‘Prime-Jim?’, ‘Shawnee’ and ‘Triple Crown’) using a detached cane assay. Ends of stem segments were sealed with wax and wound-inoculated using squares of media with mycelium. Segments were kept in humid containers on a lab bench and reaction was evaluated ten days after inoculation by measuring the area of the resulting lesion. ‘Chicaksaw’ was found to be the most susceptible by this assay, while ‘Arapaho’ and ‘Triple Crown’ were found to be the most resistant.     

Further Work on Plant Injection for Diagnostic and Curative Purposes

Summary

Randomly Amplified Polymorphic DNA (RAPD) markers were used to evaluate genetic similarity and inter-relationship among31 acid citrus species and cultivars, including sour oranges (six accessions); ‘Yuzu’ (four accessions) andits relatives (21 accessions). Out of the 60 decamer primers screened, 27 were selected which produced 108 markers; 76 of which were polymorphic. Species or cultivar-specific RAPD markers were also found. A dendrogram based on genetic distance implied that sour oranges were very distinct from ‘Yuzu’ and its relatives. ‘Yuzu’ accessions were very closely linked to each other, however; for the other specimens genetic polymorphism could easily be detected by RAPDs and the genetic variation between accessions was quite high and revealed their different origins. In this study some RAPDs allowed the distinction of very close cultivars, for instance ‘Kabosu’ from ‘Aka kabosu’.     

Determining the S-genotypes of several sweet cherry cultivars based on PCR-RFLP analysis

Summary

Based on the cDNA sequences encoding sweet cherry self-incompatibility associated ribonucleases (S-RNases), a PCR-based S-allele typing system for sweet cherry cultivars has been recently developed. Using this technique, we determined S-genotypes of the three newly released Japanese cvs Kouka-Nishiki, Beni-Sayaka and Beni-Shuho and one British cv Merton Glory that was classified as a Universal Donor, which is able to be used as a pollen donor for all cultivars in pollen incompatibility groups I to XIII. Furthermore, we also determined the partial sequences of the S-RNase genes of ‘Rainier’ (S¹S⁴)‘ and ‘Sato-Nishiki (S³S⁶)’,which leads to the development of a more reliable S-allele identification method of PCR-RFLP for sweet cherry cultivars. Total DNA isolated from leaves of the four cultivars along with those from ten cultivars with known S-genotypes were PCR amplified with two sets of primers that were designed from DNA sequences encoding the signal peptide (Pru-T2) and two conserved domains (Pru-C2 and Pru-C4R) of sweet cherry S-RNases. By comparing the size of PCR products on agarose gel, the 5-genotypes of ‘Kouka-Nishiki’, ‘Beni-Sayaka’, ‘Beni-Shuho’ and ‘Merton Glory’ were suggested to be S¹S³, S¹S⁶, S⁴S⁶, and S⁴S⁶, respectively. Two of these three S-genotypes (S¹S⁶ and S⁴S⁶) were found for the first time. DNA sequencing of PCR products from S-alleles of ‘Rainier’ and ‘Sato-Nishiki’ revealed that Ban II, Nru I, Apa LI and Ava I sites, respectively, were unique in the S¹-, S³-, S⁴- and S⁶- sequences flanked by Pru-T2 and Pru-C4R primers. RFLP analysis of the PCR products using these enzymes confirmed that S¹-, S³-, S⁴- and S⁶-alleles of the four cultivars contained the respective restriction enzyme recognition sites.     

Molecular typing of rose cultivars using RAPDs

Summary

A rapid molecular typing method is described in this work. RAPD amplification products are very dependent upon various factors such as source of taq DNA polymerase, thermocycling programmes and DNA concentrations. This issue has been consequently been examined to establish a suitable experimental protocol. Subsequently, genomic DNA from 25 cultivars of rose were amplified using RAPD techniques with twenty 10-mer primers (Operon Kit A). The data obtained reveal no variability within cultivars and a high degree of variation between cultivars. With the patterns obtained with two of the primers (OPA-11 and OPA-17) all the rose cultivars were unequivocally identified. The results suggest that RAPD profiles provide a simple and efficient way to identify rose cultivars.     

Self- and cross-(in)compatibility between important apricot cultivars in northwest Iran

Summary

Knowledge of the self-(in)compatibility trait in commercial apricot cultivars is of great importance for breeders and growers. Five commercial apricot cultivars, widely grown in Iran, were self- and cross-pollinated to determine their pollen and stylar compatibility. Fruit-set in the orchard and pollen tube growth in pistils, from flowers pollinated in the laboratory, were evaluated. In addition, specific primers previously designed to amplify fragments of the S alleles responsible for the incompatibility trait, were used to amplify DNA extracted from the five cultivars.All results agreed and confirmed that three out of the five cultivars studied were self-incompatible, two of which were cross-incompatible and therefore had the same genotype. The cultivars, ‘Ghorban-e-Marageh’ and ‘Ghermez-e-Shahroodi’ were self-compatible and, interestingly, shared a PCR band with all Spanish self-compatible apricot cultivars examined to date.     

Development of SCAR markers to differentiate between mume (Prunus mume Sieb. et Zucc.) and apricot (P. armeniaca L.)

Summary

Mume (Prunus mume Sieb. et Zucc.) and apricot (P. armeniaca L.) are similar in fruit and tree morphology, and exhibit high cross- and graft-compatibility with each other. It is therefore difficult to differentiate mume and apricot cultivars on the basis of morphological and phenotypical characteristics. Molecular markers were developed to differentiate nine mume from ten apricot cultivars. Four dominant, random amplified polymorphic DNA (RAPD) markers that can discriminate between mume and apricot cultivars (designated OPA15₆₂₈, OPO10₅₅₀, OPO20₂₅₉, and OPU03₄₁₅) were identified from 21 decamer primers. Two RAPD markers (OPO10₅₅₀ and OPU03₄₁₅) were developed into dominant sequence-characterised amplified region (SCAR) markers (SCO10 and SCU03). These SCAR markers could differentiate between all mume and apricot cultivars.     

Assessment of genetic relatedness among mango cultivars of India using RAPD markers

Summary

DNA-based RAPD (Random Amplification of Polymorphic DNA) markers have been used extensively to study genetic relationships in a number of fruit crops. A wide genetic diversity exists in the mango fruit in India. Present day commercial cultivars originated mainly from this subcontinent. In this study, 18 commercial mango cultivars, traditionally grown in western, southern, northern and eastern parts of India, were selected to assess genetic relatedness. Total genomic DNA was extracted and subjected to RAPD analysis using 30 arbitrary 10-mer primers. Of these, 27 primers amplified mango genomic DNA. None of these primers produced unique band pattern for each cultivar. RAPD data were used to calculate a squared Euclidean distance matrix, and based on this cluster analysis was done using a minimum variance algorithm. Cluster analysis clearly showed two groups—the first consisting of western, northern and eastern mango cultivars and the second group consisting of southern cultivars. From the analysis of results, it appears the majority of mango cultivars originated from a local mango genepool and were domesticated later.     

Identification of persimmon (Diospyros kaki) cultivars and phenetic relationships between Diospyros species by more effective RAPD analysis

Randomly amplified polymorphic DNA (RAPD) markers were used to identify cultivars of persimmon (Diospyros kaki Thunb.) and to estimate the phenetic relationships between Diospyros species. Long random primers (15 and 20 bases) were used to increase the effectiveness of recognizing polymorphism. Twenty-five of 34 (74%) long random primers amplified polymorphic bands of 25 persimmon cultivars, indicating that the effectiveness improved compared with RAPD analysis using 10-base primers. The 25 primers amplified a total of 444 polymorphic bands, which distinguished all 25 cultivars. RAPD markers (133) clarified the phenetic relationship between the six species and subspecies of Diospyros: D. lotus, D. lotus subsp. glabbra and D. taitoensis formed one group (lotus group), and persimmon was related in order of closeness to the lotus group, D. oleifera, and D. rhombifolia.     

Fruit Quality and Antioxidant Properties in Alabama-Grown Blackberries During Fruit Maturation

Abstract

Changes in fruit quality attributes and antioxidative properties from six cultivars of thornless blackberries (Rubus sp.) (‘Apache’, ‘Arapaho’, ‘Chester’, ‘Loch Ness’, ‘Navaho’, and ‘Triple Crown’) during four different ripening stages (red, motded, shiny-black, and dull-black) were determined under Alabama growing conditions. Berry fruit samples were evaluated for pH, titratable acidity, total soluble solids, TSS/TA ratio, soluble sugars, vitamin C (reduced, oxidized, and total) and antioxidant capacity (measured as trolox equivalent antioxidant capacity, TEAC). Significant variation among cultivars and maturity of harvest were in fruit quality attributes and antioxidative properties found. An increase in fruit pH concomitant with a decline in titratable acidity (TA) was observed during ripening for all cultivars. Total soluble solids (TSS) values increased from 5.7 to 11.6%, and TSS/TA ratio ranged from 11.9 to 63.6. Highest reducing and total sugar content were contained in dull-black fruit. Vitamin C content either declined or remained unchanged with ripening and the pattern was dependent on cultivar, maturity at harvest. In general, antioxidant activity declined between red and dull-black ripening stages. The results indicate that TSS/TA ratio and TEAC were good indicators of fruit maturity and nutritional quality, respectively.     

Cross-(in)compatibility in apricot (Prunus armeniaca L.)

Summary

Eight apricot (Prunus armeniaca L.) cultivars and selections were self- and cross-pollinated in order to determine their pollen and stylar compatibility. Overall, 40 pollination combinations were examined. Pollen-tube growth in pistils pollinated in the laboratory was analysed using fluorescence microscopy. Three inter-incompatiblity groups of cultivars were found, of which two had not been described previously, while an existing group was expanded with one additional cultivar. The first group consisted of three Hungarian cultivars (‘Ligeti Orias’, ‘Cegledi Orias’, and ‘Szegedi Mammut’) and a Moldavian cultivar (‘Kostjuzhenskyi’). The second group consisted of two American cultivars (‘Stark Early Orange’ and ‘Nugget’). The third group consisted of two Serbian selections (‘Novi Sad Early’ and ‘Frushka Gora Early’). In the incompatible cultivar combinations, pollen-tube growth stopped in the style with the formation of the characteristic swelling. In the compatible combinations, the pollen tubes reached the ovary in the majority of the pistils examined.     

Development of ISSR- and RAPD-derived SCAR markers for identification of Gladiolus germplasm

Gladiolus is an economically important ornamental crop, cultivated for its beautiful flowers throughout the world. The correct genotype identification of plant material is very significant for the floriculture industry. The aim of this study was to develop sequence-characterised amplified region (SCAR) markers from RAPD and ISSR fragments for identification and authentication of Gladiolus germplasm. The SCAR markers developed could be easily employed as valuable tools to identify newly developed Gladiolus cultivars. The SCAR markers, viz. ScG12, ScG34, and ScG36, are specific to the DNA from all 62 Gladiolus cultivars, as they did not amplify the DNA of other taxa of the family Iridaceae, including Iris, Amaryllis, and Narcissus. All three SCAR markers distinguished Gladiolus from other taxa of the family Iridaceae, whereas marker ScAm was specific to the ‘Amethyst’ cultivar. Our results confirmed that this particular SCAR marker distinguished the ‘Amethyst’ cultivar from the other 62 Gladiolus cultivars investigated in the present study. This development of SCAR markers based on RAPD and ISSR markers seems to be the maiden attempt for Gladiolus cultivars.     

Monitoring the genetic fidelity of micropropagated plantlets of Spondias mangifera Willd. using RAPD marker assays

Summary

Random amplified polymorphic DNA (RAPD) markers were used to screen for clonal fidelity in in vitro-propagated plantlets of Spondias mangifera produced through direct organogenesis. One micropropagated plantlet was selected at random after each sub-cultural passage (six sub-cultures), along with the donor plant, for RAPD marker analysis. Twenty-five RAPD primers were used to study genetic similarities or dissimilarities with the mother plant as well as among the regenerated plants. Individual primers showed that the same pattern of RAPD markers was shared by all in vitro-propagated plantlets and the mother plant. No variation was observed among the micropropagated progenies. Thus, in vitro-regenerated plantlets of S. mangifera were clonally uniform and genetically stable.     

Stilbene Levels in the Tissue and Juice of Muscadine Grapes (Vitis rotundifolia Michx.)

Abstract

Stilbenes are secondary metabolites of a class of non-flavanoid phenolics found in certain species of Vitis. This class of compounds includes resveratrol and a resveratrol glucoside called piceid that exists in trans and cis forms. This study was undertaken to determine the cis and trans forms of both resveratrol and piceid in the skin, pulp, seeds, and juice of nine cultivars of muscadine grapes and three cultivars of Vitis labruscana. Juice samples of the muscadine cultivars ‘Fry’, ‘Hunt’, ‘Magnolia’, ‘Watergate’, ‘Carlos’, ‘Noble’, and ‘Sweet Jenny’, and the V. labruscana cultivars ‘Albermarle’, ‘Miss Blanc’, ‘MidSouth’ and ‘Miss Blue’ were harvested at the full ripe stage and divided into two sub-samples. One subsample was used to extract juice and the other was divided into skins, pulp, and seeds. Sample analysis was performed using HPLC with a UV detector. Sample chromatagrams were compared with those of known standards for quantification. ‘Carlos’ and ‘Magnolia’ had the greatest total stilbene concentration in skin tissue and had bronze skin containing higher concentrations of total stilbenes than any of the black-skinned cultivars tested. Only one bronze skin cultivar, ‘Sweet Jenny’, had lower stilbenes than the dark skin cultivars. Piceid, cis piceid and resveratrol were found in the skins of all cultivars. ‘Carlos’ ‘Magnolia’, ‘Fry’ and ‘Albermarle’ muscadines contained cis resveratrol in the skins of the fruit. ‘Watergate’ was the only cultivar in which resveratrol was detected in seed tissue. ‘Sweet Jenny’ had the highest levels of cis piceid in seed tissue. ‘Albermarle’, ‘Carlos’ and ‘Sweet Jenny’ had significantly greater levels of cis picied in pulp tissue than all other cultivars.     

Sexual compatibility within and between olive cultivars

Summary

Self- and cross-incompatibility of the olive cultivars Frantoio, Manzanillo, Kalamata, Pendolino, and Picual were investigated using a 5 × 5 diallel matrix. Pistils were collected seven days after controlled pollinations on the day of flower opening, and pollen tubes were detected by fluorescence microscopy. Diallel analysis showed significant specific combining ability, general combining ability and reciprocal effects between cultivars for pollen tube growth in the pistil. ‘Frantoio’ was cross-compatible, as either a male or female parent, with each of the other cultivars, but showed a high degree of self-incompatibility. ‘Manzanillo’, ‘Kalamata’, ‘Pendolino’, and ‘Picual’ were crossincompatible, and all except for ‘Manzanillo’, were self-incompatible. It is concluded that ‘Frantoio’ is a good general polleniser for the other cultivars investigated. Pollen tube growth decreased in discrete steps from stigma to upper style, and from upper style to lower style, with the result that only one, and rarely more, pollen tube penetrated ovules. The sex ratio of flowers, and pollen viability using fluroescein diacetate staining and in vitro germination, were examined. ‘Frantoio’, ‘Manzanillo’ and ‘Pendolino’ had more than 80% perfect flowers, while ‘Kalamata’ and ‘Picual’ had less than 30%. ‘Frantoio’ had the highest pollen viability, ‘Kalamata’ and ‘Picual’ were intermediate, and ‘Manzanillo’ and ‘Pendolino’ the lowest. Pollen staining and both in vitro and in vivo germination provided the same male fertility rankings of cultivars.     

Morphology and anatomy of pollen grains from male-fertile and male-sterile cultivars of chestnut (Castanea sativa Mill.)

Summary

Pollen morphology and ultrastructure are described for four male-fertile cultivars (‘Firdola’, ‘Karamehmet’, ‘Sar?a?lama’, and ‘Hac?ömer’) and two male-sterile cultivars (‘Osmanog?lu’ and ‘Vakit Kestanesi’) of chestnut (Castanea sativa Mill.) using light microscopy and both scanning and transmission electron microscopy. Pollen grains of both male-fertile and male-sterile chestnut cultivars are tricolporate, the germinal furrow extending almost the full length of the grain axis. Pollen grains have a slightly reticulate exine. Pollen grain length varied from 13.33 – 21.30 µm, and decreased significantly in the male-sterile cultivars. Three different pollen shapes were observed among the cultivars: prolate, perprolate, and sub-prolate. The ultrastructure of the pollen grains did not differ between male-fertile and male-sterile cultivars. Intine, exine and total wall thickness (exine + intine) of pollen grains were determined as: 83.2 – 153.1 nm, 432.8 – 520.0 nm, and 516.0 – 651.6 nm, respectively; and variations were significant (P ≤ 0.05) among cultivars. The percentages of in vitro germination of pollen grains of male-fertile cultivars were between 11 – 78%, and the variations among cultivars were significant (P ≤ 0.05). The percentages of empty pollen grains observed among cultivars ranged from 3 – 32%. The correlation coefficient between the percentage of normal pollen and the germination rate was r = 0.898 (P ≤ 0.01).     

Determination and confirmation of S-RNase genotypes of apple pollinators and cultivars

Summary

We analysed the S-RNase genotypes of 23 crab apple (Malus spp.) pollinators and 102 cultivars of domestic apple (Malus pumila Mill.) by PCR amplification and digestion. Within the 23 pollinators, four pollinators, ‘Hopa’, ‘Jack’, ‘Pink Perfection’ and ‘Profusion B’, each had two unidentified S-RNase alleles. These cultivars should be useful pollinators for all domestic cultivars. Twenty-one of the domestic cultivars exhibited S-genotypes contrary to those expected from their supposed parentage, suggesting that one or both reported parents were wrong. We confirmed many of the S-RNase genotypes by pollination tests.