Analysis of tissue culture-derived variation in pea (Pisum sativum L.)-preliminary results

Summary The possibility of producing agronomically-useful somaclones via organogenesis and somatic embryogenesis from callus cultures of pea (Pisum sativum L.) was studied. Organogenic calli were induced from immature leaflets on MSB medium with NAA and BAP. Embryogenic calli were derived either from immature zygotic embryos (using 2,4-D) or from shoot apices (using picloram) of aseptically-germinated seedlings.The seed progenies (T₁ to T₃-generation) of primary regenerants were grown in field conditions and their phenotypic variation was evaluated and compared with control, non-tissue culture-derived plant material. In addition, electrophoretic analyses of selected isoenzyme systems and total proteins have been done. The results do not show dramatic changes in qualitative and quantitative traits. The evaluation of at least two future generations (T₄, T₅) is planned.Abbreviations BAP 6-benzylaminopurine - IBA indole-3-butyric acid - MSB medium (mineral salts after Murashige & Skoog, 1962, vitamins after Gamborg et al., 1968) - NAA -naphthalene-acetic acid, picloram-4-amino-3,5,6-trichloro picolinic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - ORG organogenesis - SE somatic embryogenesis     

Assessment of genetic variability in grapefruits (Citrus paradisi Macf.) and pummelos (C. maxima (Burm.) Merr.) using RAPD and SSR markers

The genetic variability of 38 grapefruit (Citrus paradisi Macf.) and three pummelos (C. maxima (Burm.) Merr..) accessions was evaluated using RAPD, and single sequence repeat (SSR) analyses. Approximately49% of the 198 RAPD were polymorphic, and 4.6 alleles per SSR loci were identified. PIC values changed from 0.093 to 0.450. A UPGMA phenetic tree was constructed and two main grapefruit groups were identified. The grapefruit accessions `do Cabo' and `Siamesa-Filipinas'clustered very close to the pummelos in Group A. The Group B consisted of three sub-groups, which comprised all of the other grapefruit accessions. The majority of grapefruit accessions showed a narrow genetic base suggesting that the observed morphological polymorphism within the group must be associated with somatic mutations, which were not detected by these molecular markers. This revised version was published online in August 2006 with corrections to the Cover Date.     

Analysis of genetic diversity in Agave tequilana var. Azul using RAPD markers

By federal law in Mexico, A. tequilana Weber var. Azul is the only variety of agave permitted for the production of any tequila. Our objective was to assay levels of genetic variation in field populations of A. tequilana var. Azul using randomly amplified polymorphic DNA (RAPD) markers. Ten plants were collected from each of four different fields, with two fields being located in each of two principal regions of Mexico for the cultivation of A. tequilana var. Azul. The two regions are separated geographically by approximately 100km. Genetic relationships between A. tequilana var. Azul and two other varieties of A. tequilana Weber, ‘Chato’ and ‘Siguin’, were also investigated using RAPDs. Among the three varieties, 19 decamer primers produced 130 markers, of which 20 (15.4%) were polymorphic betweenA. tequilana var. Chato and A. tequilana var. Siguin. The results of RAPD analysis suggest that A. tequilana var. Siguin is more closely related to A. tequilana var. Azul than is A. tequilana var. Chato. Among the 40 field selections of A. tequilana var. Azul, only 1 of124 RAPD products (0.8%) was polymorphic and 39 of 40 plants were completely isogenic. This is one of the lowest levels of polymorphism detected to date for the analysis of a crop species, and is proposed to be the result of the promotion of a single conserved genotype over many years due to an exclusive reliance on vegetative propagation for the production of new planting materials. The significance of these results is discussed in relation to breeding programs focused on the improvement of A. tequilana var. Azul. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Somaclonal variation in sugarcane through tissue culture and evaluation for quantitative and quality traits

Eight genotypes of Saccharum officinarum were crossed with Saccharum spontaneum and 14 genotypes of S. officinarum were crossed with Erianthus arundinaceus. A total of 39 hybrids were evolved. These 39 hybrids were raised in the field and used as donor clones for in vitro culture studies. Plantlets were regenerated from 1-month-old callus. The grown up plants were transplanted to well prepared field, to study the variations generated for the biometric as well as for biochemical characters. There were significant differences between the donor clones and their sub clones for all the character of interest. The somatic segregation was gradual and wider, showing a range of divergence from the mean towards the end of the scale. Fifty-one sub clones were selected with commercial potential which have 13% fibre, 200 cm stalk length, 10 cm internode length and pure obtainable cane sugar per cent of 10.     

RAPD analysis of genetic diversity among clones of the Ethiopian crop plant Ensete ventricosum

Random amplified polymorphic DNA (RAPD) was used to estimate genetic variability among 111 different clones of Ensete ventricosum, cultivated at 9 different collection sites in southwestern Ethiopia. PCR conditions were optimized for the amplification of DNA fragments in this crop. Out of 126 oligonucleotide primers initially screened,12 were chosen that together generated 97 reproducible polymorphic bands. Each clone proved to have a unique DNA profile, in spite of 8 duplications among the vernacular names represented in this material. The high level of genetic variability encountered is in good accordance with an outcrossing breeding system, and is also indicative of gene flow from wild enset and possibly from otherEnsete species. Genetic variation within collection sites was relatively high, with values for the Shannon-Weaver diversity index ranging from 0.44 to 0.55. A PCA (principal component analysis) demonstrated some differentiation among sites but also considerable overlapping. Partitioning the total variability at different levels with the Shannon-Weaver index shows that only14% was attributable to differences between sites and 86% to variation within sites. Pairwise comparisons of the 9 sites yielded values for Nei's unbiased genetic distances that ranged from 0.054 to 0.225.Cluster analysis as well as PCO (principal co-ordinate analysis) showed that Bonga and Chencha form a nucleus within a loose cluster, which also comprises Wolkitae, Worka, Shonae and Wondo. The remaining three sites, Answae, Setunae and Seltae, take quite isolated positions. Estimates of genetic distances did not correspond with geographical distances between collection sites. Genetic distances, as well as amount of within-site diversity, may instead be connected with the pattern of distribution for various ethnic groups in the enset-based agricultural system, and their dependency on enset as a staple food. This study is the first of its kind on enset. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evaluating genetic variation and relationships among two bromegrass species and their hybrid using RAPD and AFLP markers

The two most widely grown bromegrass species in North America are smooth bromegrass (Bromus inermis Leyss.), a hay type grass, and meadow bromegrass (Bromus riparius Rehmann), a pasture type grass. Recently a hybrid bromegrass population between the two species has been produced as a dual-purpose hay-pasture grass. Molecular markers have the potential to improve selection procedures to enhance bromegrass breeding. The objective of this study was to use RAPD and AFLP markers to determine genetic relationships and variations among bromegrass populations. Forty-three RAPD markers from 21 primers and 83 AFLP markers from seven primer combinations were used. Both marker types were able to group the individuals into their respective populations. The relationships among the individuals within each of the populations were not similar between the two marker types. Analysis of molecular variance (AMOVA) detected greater within-population variation than among-population variation for both marker types. The highest variation was observed in the hybrid population followed by meadow and then smooth bromegrass. The inter-population distance from both markers indicated that the highest genetic distance was between meadow and smooth bromegrass and lowest between smooth and hybrid bromegrass, which reflect the breeding history of the hybrid population. This study showed that both markers are capable of differentiating bromegrass genotypes into their respective populations, detecting genetic variation and relationships of the populations. Results of this study suggest that these two markers can be used in the future to enhance the current breeding practices in bromegrass, however, AFLP markers would be the marker of choice due to the high number of polymorphic markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Symbiotic variability in Vicia faba. 2. genetic variation in Vicia faba

Summary Eight varieties of Vicia faba were examined in association with a standard strain of Rhizobium leguminosarum and also with the application of mineral nitrogen. Large differences in dry matter yield, nitrogen percentage and total nitrogen uptake were apparent between varieties and between rhizobium and mineral nitrogen treatments; the interaction was also significant. Genotypic differences in both the effectiveness of the symbiosis with this strain of rhizobium and the efficiency of utilization of nitrogen were thus demonstrated. The implications of this variability on the assessment of varieties and the possiblity of exploiting it by plant breeding techniques are discussed.     

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     

Genetic relationship of Mediterranean mandarins (Citrus deliciosa Tenore) using RAPD markers

Summary Random amplified polymorphic DNA (RAPD) analysis was carried out to evaluate polymorphism and genetic similarity between 39 Mediterranean mandarin genotypes. One hundred eleven amplification products were identified using 21 random primers. An average of 2.2 RAPD markers was obtained for each primer, corresponding to 42% of the amplification products. Genotype-specific RAPD markers were also found, mainly in known hybrids. UPGMA cluster analysis revealed the low level of genetic variation between accessions of Mediterranean mandarins, whereas their hybrids with other Citrus species showed greater genetic dissimilarity. Twenty accessions yielded very similar patterns, suggesting either that they could be a single clone, or that the technique was not able to detect genomic variation. However, for the other specimens genetic polymorphism can easily be detected by RAPD, although the genetic variation between accessions was quite low. The large number of hybrids and the low polymorphism between accessions support the hypothesis that Mediterranean mandarins are all true hybrid of Common mandarins (Citrus reticulata Blanco).     

Genetic variability measures among Bromus catharticus Vahl. populations and cultivars with RAPD and AFLP markers

The objectives of this study were to optimize RAPD and AFLP techniques in B. catharticus, and to determine the genetic variability of populations and commercial prairie grass cultivars with the aforementioned molecular markers. Two populations with contrasting morphological characteristics were evaluated from individual and bulked DNA samples using RAPD markers. Both analyses showed a similar information about inter population variability. Each accession was sampled by a single leaf bulk of 10 plants. Accession similarities were established with 276 RAPD and 714 AFLP bands using Jaccard similarity coefficient. The dendrogram of the accessions using RAPD markers showed that they shared high similarity values (>94%). A similar result was obtained with AFLP markers (similarity values >98%), revealing a narrow genetic basis in the analyzed accessions. Consequently, molecular characterization of germplasm should be considered in addition to morphological criteria, to choose the parental genotypes for breeding programs of this forage crop. The AFLP technique was more efficient to detect DNA polymorphism in our experiments and unique fingerprints were detected for all the accessions. RAPD is a simple and non expensive technique, suitable to estimate genetic similarity. This revised version was published online in August 2006 with corrections to the Cover Date.     

RAPD variability in rice (Oryza sativa L.) plants derived from desiccation-tolerant calli

Genetic modification from selfed progenies of 18 rice (Oryza sativa L.) plants regenerated from callus tissues which survived desiccation, were investigated at the DNA level using the random amplified polymorphic DNA (RAPD) method. Twelve 10-mer random primers were used to amplify DNA of progenies from the regenerated plants, and a total of 228 PCR products and 1780 DNA fragments were obtained by primers, generating between four to thirteen major bands. The size of the amplified fragments ranged from 0.2 to 2.55 kb. The results showed that 10 out of 12 primers produced polymorphic bands, two primers (RA31 and RA185) showed no polymorphism among plants tested. A dendrogram of the genetic distance was constructed based on their polymorphism, demonstrating that somaclonal variation exists in rice plants regenerated from callus which survived the desiccation treatment. Part of this variation can be useful in rice breeding. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Symbiotic variability in Vicia faba. 3. Genetic effects of host plant,rhizobium strain and of host × strain interaction

Summary Six Vicia faba populations were grown in all possible combinations with six Rhizobium leguminosarum strains in order to estimate the relative importance of the three genetic components of symbiotic variability. Additive genetic effects of host genotype and rhizobium genotype accounted for only 8.9% and 11.8% of the total phenotypic variation. Non-additive variation attriabutable to specific host genotype × rhizobium genotype interactions was by far the largest component of variation and accounted for 73.8% of phenotypic differences. Therefore the greatest improvement in symbiotic nitrogen fixation is likely to arise from simultaneous selection of both symbionts.     

A comparative analysis of the genetic relationships between rye cultivars using RFLP and RAPD markers

Summary The genetic similarities of eight closely related rye cultivars were estimated using two molecular marking techniques: restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD). Cultivars were evaluated for variation by 11 random cDNA and genomic clones used in combination with four restriction enzymes and 40 decamer primers. A total of 53 polymorphic RFLP fragments and 94 polymorphic RAPD fragments were observed. Based on the presence/absence of fragments, two genetic similarity matrices were calculated which were then used in cluster analysis. Differences between pair of cultivars were observed in RFLP and RAPD dendrograms. RFLP analysis produced estimates of genetic relationships more in accordance with the partially known pedigree of the cultivars than did RAPD analysis. The use of bulk samples of DNA in these analyses affected the sensitivity of RAPD assays more strongly. Dendrograms which took into account all fragments produced, either by RFLP or RAPD, reflected better the relationships between cultivars than did dendrograms based on only one type of marker. This reflects the importance of the number of markers used in determining the genetic relationships between genotypes.     

Assessment of genetic relatedness of the genera Ananas and Pseudananas confirmed by RAPD markers

Genetic relationships among 18 accessions, including 16 of Ananas and two of Pseudananas, were investigated using RAPD molecular markers. The procedure for DNA extraction was adapted from the method of Dellaporta et al. (1983) where an incubation in proteinase K and a purification step were included. From the total of 148 markers scored,132 (89.2%) were polymorphic. The similarity matrix was used for cluster analysis. The phenogram developed from the RAPD bands showed that for most of the cases, the accessions within a species grouped together. Nevertheless, a moderate infraspecific genetic variation was observed. For example, DNA data grouped all A. comosus accessions with a mean similarity coefficient of 0.85. Comparable results were obtained with all other species investigated. The highest genetic divergence was found withinA. lucidus where the mean similarity coefficient among accessions was0.75. A similar level of genetic polymorphism was observed among species,therefore, a definition about which species were involved in the constitution of A. comosus genotypes was not possible. These results agree with the breeders standpoint suggesting that all Ananas species belong to the primary gene pool of pineapple. This revised version was published online in July 2006 with corrections to the Cover Date.     

RAPD analysis of sporting and chimerism in chrysanthemum

Summary The potential of colchicine and the microtubule depolymerizing herbicides trifluralin, oryzalin, and amiprophosmethyl (APM) for in vitro chromosome doubling during B. napus microspore culture was studied. Colchicine was administered during the first 6, 12 or 24 h of culture with 8 different concentrations up to 3 mM, and herbicides at 6 different concentrations up to 30 M for 12 h.Treatments with moderate concentrations of colchicine (3–100 M) produced a small increase in embryo production, while concentrations above 300 M were toxic. Colchicine treatment for 12 h resulted in higher embryo production than treatment for 6 and 24 h. Duration of treatment and concentration of colchicine both had a significant effect on the chromosome doubling. The highest diploidization rates (94% diploid regenerants) were seen after 24 h treatment with 1 mM colchicine.All three herbicides were similar to colchicine in terms of their effect on embryo formation and chromosome doubling comparable to the one of colchicine, but at concentrations approximately 100 times lower. APM was less toxic than trifluralin and oryzalin, but no significant difference in chromosome doubling efficiency was detected between the compounds. The 12 h treatment resulted in a maximum of approximately 65% diploid regenerants with all three herbicides, but APM may have an advantage because of its less toxic effects. Prolonged treatment with APM (20–24 h) may produce 95–100% diploid regenerants.Abbreviations APM amiprophos methyl - DMSO dimethyl sulfoxide     

The taxonomic characterisation of annual Beta germplasm in a genetic resources collection using RAPD markers

Summary Annual beets in the genus Beta section Beta represent an important genetic resource. Representative accessions of annual beets from a beet germplasm collection were analysed using RAPD to assess the patterns of variation and relationships among them. Using arbitrary primers, markers showing variation across accessions were identified. A dendrogram of similarity was produced using these molecular markers. All the accessions analysed were classified into three major groups corresponding to species or subspecies macrocarpa, adanensis and maritima. Macrocarpa was shown to be the most divergent group in this section. Using RAPD molecular markers, it was possible to ascribe an accession to one of three taxonomic groups and overcome much of the confusion encountered when morphological traits are used for identification. The group of maritima was found to be more polymorphic than either the group of macrocarpa or adanensis at both accession and subspecies levels.     

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.