RFLP analysis of Hypericum perforatum L. somaclones and their progenies

Three adjacent EcoRI fragments of the rDNA unit from Lycopersicon esculentum, eleven anonymous genomic and two anonymous cDNA clones from Brassica napus and three restriction endonucleases: BamHI, EcoRI and EcoRV were used for RFLP analysis of the genome of Hypericum perforatum L. A polymorphic band identified with EcoRI and two rDNA probes in five somaclones originated from the same genotype was detected in all progenies of two somaclones indicating the inheritance of the molecular changes. rDNA unit heterogeneity represented by two types of RFLP pattern revealed among somaclones and seed-derived control plants using EcoRV and two rDNA probes may indicate an alloploid origin of Hypericum perforatum. The occurrence of the identical RFLP patterns in some R₀ somaclones and seed-derived plants and their progenies may be related to the apomictic mode of reproduction which is assumed to be prevalent in Hypericum perforatum. The differences in RFLP patterns of progenies when compared with the maternal plants (1 out of 10 progenies of one control plant and 1 out of 8 progenies of one somaclone) may indicate that some progenies have originated through sexual recombination. This revised version was published online in July 2006 with corrections to the Cover Date.     

Rubus coreanus as donor of resistance to cane diseases and mildew in red raspberry breeding

Summary Strong resistance to the cane diseases Elsinoë veneta, Didymella applanata and Botrytis cinerea, and to Sphaerotheca macularis, occurred in F₁ and BC₁ derivatives of an accession of Rubus coreanus. Resistance to cane spot (E. veneta) was polygenic.In eight out of ten BC₁ progenies, average grades for cane spot infections were significantly higher in white-flowered (an ₁) than pink-flowered (An ₁) plants and in hairy-caned (H) than in glabrous (h) seedlings. It is postulated that in R. coreanus factors controlling resistance are linked with An ₁ and. probably, with h.Average grades for spur blight (D. applanata) were significantly higher in white-flowered plants in nine out of ten BC₁ progenies. Spininess (S) was associated with greater susceptibility in six out of eight BC₁ families, although this difference was not statistically significant.Plants with the phenotype hAn ₁ on average provided the best source of resistance to both cane spot and spur blight.     

Minisatellite DNA ‘fingerprints’ can distinguish Rubus cultivars and estimate their degree of relatedness

Summary Minisatellite DNA from 13 Rubus cultivars, including five raspberries, two R. flagellaris derivatives, three R. ursinus derivatives, and the three raspberry-blackberry hybrids Boysen, Logan Thornless and Young, was hybridized with the M13 probe yielding cultivar-specific DNA fragment profiles. Estimates of similarity, assessed from the fragment profiles, showed a strong association with the degree of genetic relatedness among the cultivars. Boysen was shown to be closely related to Austin Thornless, Logan and Young, and is thus thought to be of similar origin to Young, that is Austin Thornless x Phenomenal. While genetic relatedness of Creston with Lloyd George was suggested, Lloyd George parentage of Creston cannot be confirmed from the data obtained.     

Genetic relatedness among eastern North American blackberry cultivars based on pedigree analysis

Blackberry genetic relationships (Rubus subgenus Rubus Watson) have not been thoroughly studied. The University of Arkansas maintains one of the largest blackberry-breeding programs in the world and thus, in-depth knowledge of the cultivars released from the program and others instrumental in their lineages can aid in breeding endeavors. Pedigrees of 32 cultivars were traced to their founding clones. Genetic contribution (GC), maximum potential similarity (MPS), inbreeding coefficient, and coefficients of relationship (CR) were calculated for all genotypes. Nineteen founding clones contributed to the 32 cultivars, ranging from <1% to 19%. The top three founding clones for mean GC conferred nearly 50% to the 32 cultivars in this study, suggesting a somewhat narrow genetic base. Calculations for MPS ranged from complete similarity (1.00) to 0.00. Individual inbreeding coefficients ranged from 0.00 to 0.25. The coefficient of relationship was used as a method of determining genetic relatedness and ranged from 0.00 to 0.74. University of Arkansas cultivars had the highest MPS and CR in the study. This may be attributed to more generations of hybridization than the other cultivars. The MPS provided some comparable results to those of RFLP and RAPD molecular studies, but CR did not. The rather narrow genetic base of eastern North American blackberry cultivars does not create an imminent, significant concern for loss of diversity. However, more diverse germplasm should be infused for continuation of heterogeneity.     

Early selection for partial resistance to powdery mildew,Podosphaera leucotricha (Ell. et Ev) Salm. in apple progenies

Summary Selection of apple for partial resistance to powdery mildew in the nursery before planting trees in the orchard can only be relied upon if genotypic differences in expression do not depend on plant age. Five progenies between susceptible cv. Melrose and five partially resistant genotypes (four CPRO-DLO selections from the cross ‘Elstar × Prima’ and cv. Prima itself) were assessed for mildew resistance following natural infection in the nursery during two successive seasons. Per progeny 60 randomly taken trees were transplanted into the orchard and their mildew resistance levels were assessed for three more years. The mean mildew resistance of the progenies in the nursery was related to that of their resistant parent, but differences in mean resistance levels in the orchard were not consistent with those in the nursery. The individual correlations of resistance between nursery and orchard also varied per progeny. In general the second year's assessment in the nursery gave the best prediction of the orchard performance over three years. In conclusion, nursery selection for partial mildew resistance can be effective, but the efficiency varies between populations.     

Segregation and linkage analysis of DNA markers in microspore derived and F2 populations of oilseed rape (Brassica napus L.)

Summary The segregation of RFLP and RAPD markers was compared in two oilseed rape (Brassica napus L.) breeding populations from the cross Topas x R4, the latter being a low linolenic mutation line. A total progeny of 68 F₂ and 40 microspore derived plants were studied with 25 markers. The results indicated a significant excess of Topas alleles at five RAPD loci in the microspore derived population. This suggests that genomic regions which probably affect microspore culture ability do not have identical distribution in the two population types.     

Pollen and coenocytic microspore germination in male-fertile and malesterilemsl soybean

Summary Light and fluorescence microscopy were used to study coenocytic microspore germination from male-sterile (ms1 ms1) soybean plants. Anther squashes from male-sterile plants revealed that a low frequency of natural coenocytic microspore germination occurred in male-sterile anthers of four independent lines; [ms1-North carolina (T260H),ms1-Urbana (T266H),ms1-Tonica (T267H), andms1-Ames (T268H)]. Abnormalities such as giant tubes, branched tubes, tubes with swollen areas, and multiple tubes were observed from coenocytic microspores from all four lines. The Urbana line, however, demonstrated a higher percentage of coenocytic microspore germination than did the other three lines. Flowers of the Urbana line from both malefertile and male-sterile plants, as well as gynoecia pollinated with coenocytic microspores from sterile plants, were used for in vivo studies. Pollen-tube growth appeared normal in male-fertile plants. In contrast, coenocytic microspore tubes rarely were observed in gynoecia from male-sterile plants or in gynoecia from malefertile plants that had been artificially cross-pollinated withms1 ms1 plants. Few tubes from coenocytic microspores were observed in the vicinity of the micropylar region. A low frequency of seed set was achieved in the greenhouse on Urbana male-sterile plants grown in the absence of male-fertile plants. Thus, we believe either that some gametes from coenocytic microspores are able to participate in fertilization at low frequency or that apomixis occurs inms1 ms1 plants.Joint contribution: Agricultural Research Service, U.S. Department of Agriculture, and Journal Paper No. J-12310 of the Iowa Agriculture and Home Economics Experiment Station, Ames IA, 50011 USA. Project 2471.     

Novel genotypes of the subtropical grass Eragrostis curvula for the study of apomixis (diplospory)

The aim of this work was to obtain a series of genetically related lines of Eragrostis curvula with different ploidy levels and reproductive modes, which can be used for the discovery of genes associated to diplospory. E. curvula is a widely cultivated forage grass native to Southern Africa that reproduces naturally by obligate diplosporous apomixis (asexual reproduction by seeds) of the Antennaria type. In vitro culture of immature inflorescences of the apomictic cultivar Tanganyika led to the regeneration of a diploid plant with a particular morphology (plant UNST1122). Embryo sac analysis and progeny tests determined that plant UNST1122 reproduces by sexuality. UNST1122 was cloned by dividing its tillers and a R ₁ generation was obtained from seeds after open pollination among the diploid clones. A set of 500 seeds from a diploid R ₁ plant was treated with colchicine and two plants showing a duplicated chromosome number (2n = 4x = 40) were obtained (plants UNST1112 and UNST1131). Progeny tests using RAPDs indicated that these plants reproduce also by sexuality. The availability of sexual tetraploid genotypes of E. curvula will allow the production of hybrids with new interesting combinations of agronomic traits and the developing of mapping populations segregating for diplospory. Besides, these genetically related plants with different reproduction modes constitutes an excellent model for the identification of gen(es) involved in diplosporous apomixis by mRNA profiling as well as to study the genomic rearrangements and gene expression alterations associated to changes at ploidy levels.     

The successful transfer of primocane fruiting expression from raspberry to Rubus hybrid berry

This paper describes the successful transfer of the primocane fruiting (PF) character from raspberry (Rubus idaeus) to Rubus hybrid berries following crosses between raspberry and blackberry. The transfer was achieved by doubling the chromosome complement of PF raspberry selections in culture using colchicine, and using regenerant tetraploid PF plants as the female parents in crosses with tetraploid or octoploid blackberries, or with hexaploid hybrid berries (all summer fruiting). The results show that the PF character is expressed more readily in hybrid plants where at least 50% of the chromosomes in the hybrid genome are derived from raspberry. In vitro germination of cut seeds overcame the poor germination often encountered in Rubus and ensured the rapid recovery of hybrid seedlings. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pollen and pollination experiments. VI. Heat resistance of pollen

Summary Pollen of dry apple, pear, lily and rose pollen was heated up to 48 h at a range of temperatures. About half or more than half of the pollen grains survived 48 h at 40 C, 24 h at 50 C, 8 16 h at 60 C. 4 8 h at 70 C, more than one hour at 80 C. and between 10 and 20 min at 90 C. Presumably, pollen able to withstand low humidity is also heat resistant, a property which may be usable to make pollen virus free through heat treatment and perhaps to overcome incompatibility.     

Plants regenerated from embryo cultures of an apomictic clone of Kentucky bluegrass (Poa pratensis L. ‘Baron’) are not apomictic in origin

Plants were regenerated from tissue cultures of embryos dissected from seeds that were harvested from a self-pollinated clonal selection of Kentucky bluegrass (Poa pratensis L.) ‘Baron’, an apomictic cultivar. Plants were regenerated from 35 embryo-derived callus cultures of the 3280 embryos that were plated. Flow-cytometric (FCM) and RAPD-marker analyses were performed to determine if regenerants were or were not apomictic in origin. Fifteen regenerants with a 3c DNA content were classified as arising from 2n + n aberrant embryos, which was a higher frequency than expected, based on a chi-square analysis. Of the remaining 20 regenerants with a 2c DNA content, a chi-square test showed that all could have arisen from n + n sexually-derived embryos, based on the observed segregation of n + n regenerants, which fit the expected 3:1 ratio of dominant:recessive RAPD-marker phenotypes. The apparent lack of regenerants of apomictic origin, and implications for genetic transformation and breeding of Kentucky bluegrass are discussed.     

Penetrance of creeping-rootedness in clonal progenies of lucerne and observations on underground morphology of plants differing for this character

Elaeis oleifera or ‘caiaué’, a close relative of oil palm (E. guineensis), has some agronomic traits of great interest for the oil palm genetic breeding such as slow growth, oil quality (mostly unsaturated) and disease resistance. An analysis of a Brazilian oil palm germplasm collection was carried out using RAPD (Random Amplified Polymorphic DNA) markers with the objective of understanding the genetic variation of ‘caiaué’ accessions collected in the Amazon Forest in the last two decades. A sample of 175 accessions obtained along the Amazon River Basin was analyzed and compared to 17 accessions of oil palm from Africa. Ninety-six RAPD markers were used in the analysis, of which fourteen were shown to be specific to oil palm, while twelve were specific to ‘caiaué’. Results showed that the Brazilian ‘caiaué’ accessions studied have moderate levels of genetic diversity as compared to oil palm accessions. The data allowed the establishment of similarity groups for ‘caiaué’ accessions, which is useful for selecting parental plants for population breeding. Cluster analysis showed that, in general, genetic similarities are not correlated to geographical distances, but consistent with geographical dispersal along the Amazon River network. AMOVA showed that most of the genetic variation is found within populations, as expected for anallogamous and long-lived perennial species. The study provides important information to define strategies for future collection expeditions, for germplasm conservation and for the use of E. oleifera in breeding programs. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Evaluation of the genetic diversity among elite tea (Camellia sinensis var. sinensis) accessions using RAPD markers

The diversity of 27 superior tea (Camellia sinensis var. sinensis) accessions from Korea, Japan and Taiwan was examined with RAPD-PCR (Random Amplified Polymorphic DNA Polymerase Chain Reaction) markers. Out of the 50 primers screened, 17 primers generated 58 polymorphic and reproducible bands. A minimum of 3 primers was sufficient to distinguish all the 27 accessions studied. The Shannon's index used to partition diversity into inter- and intra-group, revealed that 71 percent of variability resided within groups and 29 percent between groups. Diversity was greatest within the Korean group followed by Taiwan and Japan. The relatively high diversity observed in Korea might reflect the larger genetic base of its plantations while the low diversity in Japan could be explained by the long and intensive tea selection programme in this country. A dendrogram based on the UPGMA-link method using Jaccard's distances and multivariate Factorial correspondence analysis clustered the tea accessions into two main groups, regrouping the Taiwan cultivars on the one side and the Korean and Japanese accessions on the other side. This suggests that the Taiwan tea studied here may have a different origin from that of Korea and Japan. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic hybrids between Gossypium hirsutum L. (4×) and G. davidsonii Kellog (2×) produced by protoplast fusion

Somatic hybrids were obtained from electrofused protoplasts derived from embryogenic suspension cultures of tetraploid cotton (G. hirsutum L. cv. Coker 201) and embryogenic callus of diploid wild cotton G. davidsonii. The regenerants were initially identified as hybrids by RAPD (random amplified polymorphic DNA) analysis. Subsequently, observation on chromosome counting, morphology and SSR (simple sequence repeat) confirmed the hybrid status. Cytological investigation of the metaphase root-tip cells of the regenerated plants revealed there were 74 to 84 chromosomes in the plants, close to the expected 78 chromosomes. SSR analysis revealed the regenerated plants contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the plants was intermediate between the two fusion partners. The regenerants were difficult to develop into mature plants because their roots browned and they wilted from the stem apex before forming 3 to 5 true leaves. The hybrid plants were transferred to soil by grafting in vitro onto rootstocks.     

Pollen storage in cocoyam (Xanthosoma sagittifolium (L.) Schott)

Summary The effect of different storage conditions on cocoyam pollen viability was investigated. Two levels of temperature (0° and 5°C) and four levels of relative humidity (0, 10, 20 and 30 percent) represented the storage environments.Viable cocoyam pollen could be obtained after 28 days in storage. The best storage condition was 5°C and 30% relative humidity.     

Somatic hybrids between navel orange (Citrus sinensis) and grapefruit (C. paradisi) for seedless triploid breeding

Protoplasts from cell suspension cultures of ‘Bonnaza’ navel orange (Citrus sinensis (L.) Osbeck) were electrically fused with mesophyll protoplasts isolated from seedless ‘Red Blush’ grapefruit (Citrusparadisi). After 6 months of culture, a total of 20 plants were regenerated. Root tip chromosome counting revealed that 4 of them were tetraploids (2n = 4x = 36)and the rest were diploids (2n = 2x = 18) morphologically resembling the mesophyll parent. After 6 months of transplantation into the greenhouse, 4 of the diploidmesophyll regenerants unexpectedly flowered, but this phenomenon disappeared in the next year. This is the first report of precocious flowering in citrus via protoplast fusion. RAPD analysis further confirmed that the tetraploid regenerants were somatic hybrids while the diploid regenerants were mesophyll parent type. This somatic hybrid will be utilized as a possible pollen parent for improving the seedy pummelo cultivars in China by producing triploid seedless pummelo hybrid. The mechanism of early flowering was also discussed. This revised version was published online in July 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.     

RAPD characterization of Brazilian barley (Hordeum vulgare ssp. vulgare) varieties

Thirteen varieties of Brazilian barley selected for malting qualities were analysed by RAPD (Random Amplified Polymorphic DNA). Amplification with 18 random primers generated 221 reproducible bands, of which 206 bands were polymorphic (93%):of this number, 137 fragments (62%) detected diversity among varieties and 56 bands (25.34%) allowed the distinction of varieties or groups of them. Variation was detected in all Brazilian varieties studied. Within-variety similarities estimated by Jaccard Similarity Coefficient ranged from 0.28 to 0.94, with averages ranging from 0.57 to 0.83, and an overall average of 0.72. Nevertheless, in the cluster analysis representatives of the same variety always fell into the same group and only later joined the other varieties. The average intervarietal similarities estimated by Jaccard Similarity Coefficient ranged from 0.45 to 0.62, with an overall average of 0.52. Many bands or combinations of bands which were responsible for the differentiation of all varieties were detected. Nevertheless, the majority of these bands cannot be considered as diagnostic markers because a great number of them did not occur in many representatives of the variety or had low intensity or even because they were not easily identified in the total pattern of bands.     

RAPD variation in Solanum anguivi Lam. and S. aethiopicum L. (Solanaceae) in Uganda

Variation in 39 polymorphic RAPD markerswas studied for 18 populations of Solanum anguivi, S. aethiopicum Gilogroup and S. aethiopicum Shum groupin Uganda. UPGMA and PCO analyses of thedata revealed no clear groupingscorresponding to the species and groups. AnAMOVA analysis of the genetic variationshowed that the variation among species andgroups was less than 10%, whereas thevariation within species and groups wasmore than 90%. A Mantel test showed nocorrelation between genetic distance andgeographic distance. The data gives onlyvery weak support for the separation of themore morphologically distinct S.aethiopicum and S. anguivi. TheGilo- and Shum-groups of S.aethiopicum, which were previouslyregarded as two separate species can not beseparated on the basis of RAPD data, and itis most likely that they are keptmorphologically distinct by a strongman-made selection pressure.