Monitoring genetic fidelity vs somaclonal variation in Norway spruce (Picea abies) somatic embryogenesis by RAPD analysis

Summary Somaclonal variation, which is a welcome source of genetic variation for crop breeding, is unwanted when direct regenerants have to be used in tissue culture mass propagation (eg. in many forest trees), or in the regeneration of genetically transformed plants. Random amplified polymorphic DNA (RAPD) was used to analyse somatic embryos and plants regenerated from embryogenic cell lines in Norway spruce, Picea abies (L.) Karst. RAPD facilitated the identification of clones, as material from the same cell lines shared identical patterns of amplified fragments, whereas regenerants from different cell lines were easily distinguishable by their respective patterns. For comparisons with explant donor genotypes, cell lines were initiated from cotyledons. Some of the seedlings that had parts of their cotyledons removed were grown on as control plants. Somatic embryos regenerated from cotyledon cell lines showed no aberrations in RAPD banding patterns with respect to donor plants. We conclude that gross somaclonal variation is absent in our plant regeneration system.Abbreviations ESM embryogenic suspensor mass - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism - (2,4-dichlorophenoxy)acetic acid 2,4-D - 1-naphthaleneacetic acid NAA     

Geographical distribution of genetic variation in Stylosanthes scabra revealed by RAPD analysis

A large number of S. scabra accessions have been accumulated worldwide. The majority of them were collected from Brazil and most of the others came from either Colombia or Venezuela. One hundred of these accessions, selected to represent the geographical distribution of the S. scabra collection held at the Australian Tropical Forages Genetic Resource Centre, were analysed using RAPD as markers. Seven of these accessions were found not to be S. scabra. Of the S. scabra accessions, the average dissimilarity value among Brazilian accessions (0.053) was much lower than that among Colombian (0.074) or Venezuelan (0.088) accessions, with an overall dissimilarity value of 0.059 among all the S. scabra accessions. Based on their dissimilarity values, most of these accessions could be separated into five groups. Geographical distributions for most accessions in each of these groups were well defined. Limited long distance introductions/dispersions of S. scabra between these regions were detected and they were mainly confined to Brazilian genotypes. The clustering results based on RAPD were compared with those based on morphological-agronomical characters, and the groups produced by the two different methods did not always match. Possible reasons for this discrepancy are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of genetic variation in Cucurbita moschata by random amplified polymorphic DNA (RAPD) markers

Knowledge of genetic relationships among genotypes is essential for the effective utilisation of germplasm, especially for poorly characterised species. Random amplified polymorphic DNA (RAPD) analysis provides a quick and reliable method for resolving genetic relationships. Although Cucurbita moschata Duch, also known as tropical pumpkin, is one of the most important vegetable crops in Africa, being adapted to a wide range of climatic and soil conditions, it is a scientifically neglected species. The objectives of this study were to (1) analyse the amount of genetic diversity inC. moschata landraces grown in south-central Africa and (2) classify the landraces to assist in selection of parent genotypes for improvement of fruit characteristics. Cluster analysis, based on 39 polymorphic and 105 monomorphic DNA fragments amplified by 16 primers, was used to show relationships among 31 genotypes obtained from Zambia and Malawi. The analysis revealed four clusters, with genotypes from Malawi mainly clustering in three clusters while all genotypes from Zambia and three from Malawi clustered in one cluster. The pair-wise mean genetic distance was 0.32 ± 0.04 for samples from Malawi and 0.26 ± 0.04 for samples from Zambia. The possible application of the resulting classification in breeding of C. moschata is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Suitability of RAPD analysis for the detection of somaclonal variants in oil palm (Elaeis guineensis Jacq)

Random amplified polymorphic DNA (RAPD) analysis using arbitrary 10-mer oligonucleotide primers was employed in order to investigate the genetic fidelity of somatic embryogenesis-derived regenerants of oil palm (Elaeis guineensis Jacq). Clonal palms bearing the ‘mantled’ phenotype were identified in the field and the ability of RAPD markers to distinguish these variants from palms of the normal type was assessed. Of the 387 arbitrary primers used, 259 (67%) were successfully used to amplify oil palm DNA genomic fragments with consistently reproducible banding. Of these 387 primers, 73 (19%) primers enabled the identification of polymorphism between clones. No intraclonal variability and no differences between mother palms and regenerants could be identified using the total number of markers scored (8900). Twenty-four of these 73 primers were chosen for use in a larger experiment aimed at comparing, first, the mother palm genome with that of its clonal offspring and, second, true-to-type and variant regenerants. Thus, the regeneration protocol based on somatic embryogenesis set up for oil palm clonal propagation does not induce any gross genetic changes. The results obtained revealed however, that the RAPD approach is not suitable for the detection of the ‘mantled’ variant phenotype. The use of RAPD markers for the detection of somaclonal variation in oil palm is discussed.     

RAPD analysis of genetic variation in natural populations of Betula alnoides from Guangxi, China

There is an urgent need for the developmentof early identification techniques inolive-trees due to the economic importanceof cultivar identification in periods ofexpansion like now. We have been able toidentify 22 olive-tree cultivars using only10 different, specific, repeatable markers.These markers were designed by the cloningof significant RAPD bands obtained in PCRperformed on bulked DNA to retain thegenetic variability of each cultivar.Clones were partially or totally sequencedand new primers derived from thesesequences were used to obtain SequenceCharacterised Amplified Region (SCAR)fragments. We have demonstrated that theuse of the 10 SCAR markers is enough toprovide a simple, cheap, and reliableprocedure to identify 22 geographicallyrelated olive-tree cultivars.     

Assessment of natural and induced genetic variation in Alstroemeria using random amplified polymorphic DNA (RAPD) markers

Summary We have used random amplified polymorphic DNA (RAPD) markers to study genetic variation in Alstroemeria. The first objective was to examine the discriminatory power of RAPD markers in different genotypes of Alstroemeria obtained by traditional breeding. All genotypes examined, including commercial Alstroemeria varieties, could be distinguished on the basis of their RAPD profiles. Progeny plants could be distinguished from their parents. A second objective of this study was to investigate whether RAPD markers can be used as a routine tool to detect mutant plants, as an alternative to glasshouse testing. To address this objective, we analysed Alstroemeria plants that carried phenotypically visible mutations that either were induced by irradiation using X-rays or were the result of somaclonal variation. In eight out of a total of 13 mutant Alstroemeria plants obtained after irradiation or tissue culture we detected no polymorphisms when compared to control plants that were considered to be non-mutated. Only in five of the mutant plants analysed we detected one to two polymorphisms. These results suggest that frequent genome rearrangements had not occurred in the mutant plants analysed. These results also demonstrate that the RAPD technique is an inappropriate tool for the rapid screening of Alstroemeria for induced variation. It that the RAPD technique is an inappropriate tool for the rapid screening of Alstroemeria for induced variation. It seems probable that this conclusion would be equally applicable in other plant genera in which induced variation has occurred. However, the RAPD technique is a simple and effective tool for genetic fingerprinting of Alstroemeria varieties, provided their differences are due to sexual propagation.     

Relationships in pineapple by random amplified polymorphic DNA (RAPD) analysis

Random amplified polymorphic DNA (RAPD) analysis was applied to eight commercial cultivars of pineapple, two intergroup hybrids and two wild species. Morphologically, pineapple is divided into the Cayenne, Queen, Spanish, Maipure and Abacaxi groups. Members of the first three groups have been analysed in this study. The cultivars ‘Tradsithong’, ‘Phuket’, ‘Sawee’ and ‘Tainan’, with spiny leaves, form the Queen group. In ‘Pattavia’, ‘Nanglae’ and ‘Petburi no. 2’ (Cayenne group), spines are confined to the leaf tips. ‘Intrachitdang’ is normally placed in the Spanish group, which is morphologically similar to the Queen group, but with inferior quality fruit. DNA amplification products were compared from 16 arbitrary 10‐mer primers from which a dendrogram was constructed. The results confirmed morphological classifications for seven of the eight commercial cultivars, with the Queen and Cayenne groups as separate clusters. However, the cv. ‘Intrachitdang’ was more closely related to the Cayenne group. Two hybrids from reciprocal Cayenne × Queen group crosses, were more closely allied to the Queen group. The two wild species were outside the groups. RAPD analysis can be exploited to investigate relationships within pineapple germplasm.     

The genetic diversities of 4 species of subg. Lithocerasus (Prunus, Rosaceae) revealed by RAPD analysis

RAPD analysis was applied to reveal the genetic diversities of 4 speciesof subg. Lithocerasus within the genus Prunus using 40accessions representing the subgenera Prunophora, Amygdalus,Lithocerasus and Cerasus. The accession of subg. Lithocerasus are phenotypically similar to members of subgenus Cerasus but different with them in interspecific crossing tests and isozymeprofiles. Two major clusters, `Prunophora and Amygdalus group' and `Cerasus group' were constructed in the phenogram. We revealthat the examined 4 species of subg. Lithocerasus species; P.tomentosa Thunb., P. japonica Thunb., P. glandulosa Thunb.and P. besseyi Bailey, were genetically closer to the members ofsubgenera Prunophora and Amygdalus than to subg. Cerasus.     

Genetic variation and phylogenetic relationships based on RAPD analysis in section Caulorrhizae, genus Arachis (Leguminosae)

Wild Arachis germplasm includes potential forage species, such as the rhizomatous Arachis glabrata and the stoloniferous A. pinto and A. repens. Commercial cultivars of A. pintoi have already been released in Australia and in several Latin American countries, and most of these cultivars were derived from a single accession of A. pintoi (GK 12787). Arachis repens is less productive as a forage plant than is A. pintoi. However, it can be crossed with A. pintoi, and thus has good potential as germplasm for the improvement of A. pintoi. Arachis repens is also used as an ornamental plant and ground cover. Many new accessions of these two stoloniferous species are now available, and they harbor significant genetic variability beyond that available in the few older accessions, previously available. Therefore, these new accessions need to be conserved, documented and considered in terms of their potential for crop improvement and direct commercial use. Sixty-four accessions of this new germplasm were analyzed using RAPD analysis. Most of the accessions of A. repens grouped together into a clearly distinct group. In general, the accessions from the distinct valleys of the Jequitinhonha, São Francisco and Paranã rivers did not group together, suggesting there is not a tight relation between dispersion by rivers and the geographic distribution of genetic variation in these species.     

Field evaluation of somaclonal variation in sunflower (Helianthus annuus L) and its application for crop improvement

Immature zygotic embryos from the American fertility restorer line RHA-857 were used as donor material for induction of direct organogenesis in sunflower (Helianthusannuus L.). The range of spontaneous somaclonal variation among the progenies of regenerants was studied. The genetic modifications observed in regenerants included agronomic traits such as oil content in seed, 1000 seed weight, plant height, leaf width, leaf length, petiole length, internode length, head diameter, number of branches, length of branches, number of ray florets, seed width, seed length, and seed thickness. RAPD molecular analysis carried out on sunflower materials in the R-11 generation showed the absence of a specific 358 bp band in somaclonal line 11/2/51 R. This line showed a modified architecture, full resistance to Phomopsis helianthiand higher oil content in seed in comparison to the standard RHA-857. Line31/3/53 R was with modified architecture and higher 1000 seed weight. Hybrid No. 144 produced with the participation of somaclonal line 20/5/52 R demonstrated high production capacity, shorter vegetation period and reduced height. The combination of these favourable changes is desirable in breeding work on sunflower. Somaclonal variation through director ganogenesis has facilitated the creation of genetically heritable variation in sunflower, which can be used with great success for hybrid seed production of highly productive hybrids. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic control of somatic embryogenesis in alfalfa (Medicago sativa L. cv. Adriana)

Summary In alfalfa (Medicago sativa) regeneration is genotype-specific. In order to study the genetic control of somatic embryogenesis and to constitute a synthetic cultivar characterized by its high regeneration ability, 2 embryogenic plants selected from the cv. Adriana were selfed, intercrossed and also crossed in both directions with 5 non-embryogenic genotypes of the same cultivar.Progenies of all crosses were scored for their regeneration ability and results indicate that somatic embryogenesis is under the control of 2 dominant loci. However some non-embryogenic genotypes prevent regeneration when crossed with embryogenic ones and this characteristic is not under the control of a single dominant gene.When plants chosen for their capacity to regenerate within F₁ and S₁ progenies were freely intercrossed the regeneration efficiency dropped to 2% (1 plant out of 50). This result indicates that if the genetic background of the population is changed the regeneration is greatly affected and therefore some other mechanism could play a role in determining plant regeneration.     

Use of RAPD and microsatellite (SSR) variation to assess genetic relationships among populations of tetraploid alfalfa, Medicago sativa

The level of random amplified polymorphic DNA (RAPD) and microsatellite variation present in four ecotypes and two varieties of alfalfa (lucerne) from Italian and Egyptian germplasm sources was evaluated. A sample of 100 plants from 10 populations was analysed by means of 41 RAPD markers and 37 simple sequence repeat (SSR) markers. Both molecular approaches revealed a high degree of genetic diversity within each of the cultivated populations and enabled each of the plants considered to be uniquely fingerprinted. The genetic relationships among plants and populations were analysed by computing AMOVA (analysis of molecular variance) and F_ST analyses. RAPDs were able to separate the Italian populations from the Egyptian variety. SSRs allowed strong separation of the four Italian alfalfa ecotypes. It was concluded that RAPD and microsatellites could be useful and powerful tools for assessing genetic variation and genetic relationships in tetraploid alfalfa.     

Genetic variation of mitochondrial and nuclear genomes in carrots revealed by random amplified polymorphic DNA (RAPD)

Mitochondrial and nuclear genomic diversities of 8 carrot (Daucus carota ssp. sativus) varieties, including 6 pure lines and 2 cytoplasmic male sterile (cms) lines, were taxonomically identified using PCR with 19 RAPD primers. Dendrograms based on polymorphisms of both mitochondrial and nuclear genomes were constructed. According to the dendrogram of the mitochondrial genome revealed by RAPD, 4 differentiated clusters formed, in good accordance with the classification based on analyses with restriction enzyme digestion. Two cms lines were grouped into the same cluster, as genetically separated from the others. Thus, the cytoplasm donors of these male sterile lines were thought to be wild carrots. Conversely, RAPD analysis of the nuclear genome for these eight cultivars revealed no evident clusters although some cultivars were of a similar origin or place of cultivation. A correlation between nuclear and mitochondrial dendrograms was absent. RAPD has proved to be a useful tool for identifying mitochondrial and nuclear genomes. This technique will greatly aid in promoting efficient improvement of carrots. This revised version was published online in July 2006 with corrections to the Cover Date.     

RFLP analysis of genetic variation in species of section Arachis, genus Arachis (Leguminosae)

Four A-genome species of the genus Arachis (A. cardenasii, A. correntina, A. duranensis, A. kempff-mercadoi), three B genomes species (A. batizocoi, A. ipaënsis and A. magna),the AABB allotetraploid A. hypogaea (cultivated peanut) and introgression lines resulting from a cross between A. hypogaea and A. cardenasii were analyzed by RFLP. The A genome species (cytologically characterized by the presence of a small chromosome pair ‘A’) were closely similar to each other and shared a large number of restriction fragments. In contrast, the B genome species differed more from one another and shared few fragments. The results of this study indicate that the absence of the small chromosome pair is not a good criterion for grouping species of section Arachis as B genome species, since their genome might be quite distinct from the B genome of A. hypogaea.The lowest genetic variation was detected within accessions of A. duranensis (17 accessions), followed by A. batizocoi (4 accessions) and A. cardenasii (9 plants of accession GKP 10017).The high level of genetic variation found in A. cardenasii might indicate that not all accessions of wild species of Arachis are autogamous, as reported for A. hypogaea.     

Characterization and genetic distance analysis of cassava (Manihot esculenta Crantz) germplasm from Mozambique using RAPD fingerprinting

Twenty-eight cassava genotypes from Mozambique, along with seven genotypes from Angola, Madagascar, Nigeria, Togo, Columbia, and Thailand for comparison, were fingerprinted using random amplified polymorphic DNA (RAPD) analysis. The Mozambican material represented a wide range of landraces. A total of 311 scored RAPD loci were used to calculate genetic distances between the genotypes. This revealed an average genetic distance of 3.1% between all the germplasm. The average genetic distance between the Mozambiquen genotypes was 2.7%, whilst the seven accessions from the other countries showed an average distance of 3.4%. Neighbor-joining (NJ) method cluster analysis of the genetic distance yielded a tree that did not indicate a relationship between geographic distribution and genetic diversity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Species relationships in the subgenus Ceratotropis (genus Vigna) as revealed by RAPD analysis

Summary The genetic variation among 23 accessions of 5 species in the subgenus Ceratotropis, genus Vigna, were investigated by random amplified polymorphic DNA (RAPD) analysis. A total of 404 fragments amplified with 24 primers were scored and analyzed by cluster analysis. The accessions used were separated into two main groups with an average of 70% differences. Within the main groups, five subgroups were recognized, which are in complete agreement with taxonomic species. Wild forms were always grouped with their most closely related cultivated forms and they showed variation in each species. The largest intraspecific variation was found in V. radiata (mungbean), in which wild forms (V. radiata var. sublobata) were highly different from each other and from cultivated forms. V. angularis (adzuki bean) showed the least variation and thus, was probably differentiated in relatively recent times.     

Study of the variation in the somatic embryogenesis ability of some pea lines (Pisum sativum L. and Pisum arvense L.)

Summary Thirty lines of pea were tested in vitro to evaluate their ability to produce somatic embryos. Three distinct genotypic classes were detected (strong, medium and weak). The best responses were obtained in Pisum sativum. Abnormal somatic embryos and secondary embryogenesis seem to constitute the principal obstacle to the development of these structures.     

Genetic diversity of plums characterized by random amplified polymorphic DNA (RAPD) analysis

We investigated the genetic diversity of 42 plum varieties by RAPD analysis. Twenty primers discriminated all plum varieties excepting two synonymous pairs: 'Botankyou and ‘Kelsey’, and ‘Chairn’ and ‘Tragedy’. Two North American plums, ‘Beach Plum’ and ‘Glow’, were genetically distinct from the other plums by cluster analysis. Overlaps observed between the ‘European plum group’ and the ‘Japanese plum group’, were perhaps due to intercrossing. We could also discriminate ‘Sordum’ from 'Late Sordum and ‘Bansei Sordum’, although ‘Late Sordum’ and ‘Bansei Sordum’ are thought to be derived from bud mutants of ‘Sordum’. This revised version was published online in July 2006 with corrections to the Cover Date.     

Radiomutants of chrysanthemum (Dendranthema grandiflora Tzvelev) of the Lady group: RAPD analysis of the genetic diversity

Random amplified polymorphic DNA (RAPD) markers were used to study the molecular characterization of 10 new radiomutants of chrysanthemum. The original cultivar ‘Richmond’ differed in genetic distance from its Lady group mutants. The analysis of genetic similarity indices revealed low diversity within the radiomutants. The dendrogram obtained after cluster analysis separated the new cultivars as a group that differed from the original cultivar ‘Richmond’. The Lady group cultivars, derived from one original cultivar by radiomutation, could be distinguished from each other by using RAPD markers of only a single primer or sets of two or three primers. Polymerase chain reaction analysis proved the efficiency of the RAPD method for DNA fingerprinting of the original cultivar ‘Richmond’ and its new radiomutants.     

Evaluation of genetic variation in Lachenalia bulbifera (Hyacinthaceae) using RAPDs

RAPD (randomly amplified polymorphic DNA) analyses were carried out on 21 accessions of Lachenalia bulbifera (Cyrillo) Engl. Five pre-selected primers produced an average of 88% polymorphisms. Fifteen of the 21 accessions could be identified using the five primers. In a pairwise comparison genetic distance values ranging from 0.11 to 1.08 were obtained. These values reveal a high amount of variation within the species. The genetic distance values within the tetraploid and hexaploid groups on the south coast were low, but values were high between the groups on the south coast and those on the west coast. A dendogram was constructed from the RAPD banding profiles, using UPGM cluster analysis. The dendogram clusters certain accessions together. These clusters are supported by their geographical locality and chromosome data. The hexaploid group, tetraploid group and octoploid group on the south coast are respectively clustered together. It is concluded that RAPDs can be used to assess the genetic variation at an intra-specific level in Lachenalia. This revised version was published online in July 2006 with corrections to the Cover Date.