Spatially dependent genetic diversity within and between colonies of wild raspberry Rubus idaeus detected using RAPD markers

In the Tayside region of Scotland, red raspberry Rubus idaeus exists both as extensive plantations of clonally propagated cultivars, and as wild populations that inhabit both the cultivated areas and uncultivated uplands to the north. In order to explore the genetic diversity of the wild populations and their degree of similarity to the plantation clones, individual plants in wild populations were sampled from four distinct sites along a 25-km transect northwards from the area of cultivation. The genetic material of each of 45 individuals and the commercial cv. Glen Clova were examined using six RAPD primers giving a total of 63 variable bands. Some identical banding patterns were observed, suggesting vegetative growth up to 20 m, but populations consisted mainly of genetically distinct individuals. Similarity matrices based on the marker bands showed, without exception, that plants were more similar within a site than between sites. None of the populations was closely related to the cv. Glen Clova. Although the most northerly site had the largest proportion of rare bands, there was no general trend between within-site diversity, or similarity to the cultivar, and position on the north-south line. All four sites had unique bands and bands not displayed by the cultivar. However, the genetic diversity of a site appeared to increase as the extent of the sampled area increased, implying that the genetic variation was spatially dependent. For example, maximum and minimum similarities were 100 and 80%, respectively, at interplant distances of 2 m; 100 and 60% at distances of 20 m; 85 and 55% at 200 m and 70 and 40% at 20 km.     

Interaction of endodormancy and paradormancy in raspberry (Rubus idaeus L.)

Bud break in protected Northern European raspberry crops is often poor and uneven with many of the sub-apical buds remaining in a dormant state. In order to improve bud break and therefore yields, the mechanism controlling bud dormancy must be determined. Canes of the biennial cultivar ‘Glen Moy’ were forced as isolated single nodes, trisections, or as intact canes after different lengths of cold storage chill unit (CU) accumulation in order to determine whether the buds were in an endodormant or paradormant state. The results showed that buds on the lower parts of the intact canes remained in a dormant state long after buds from higher up the intact cane and also the single nodes from all parts of the cane had emerged from the deepest phase of endodormancy. This would imply that these buds were being held in a paradormant state until large amounts of chilling units (>1000 CU) had been accumulated. The trisected cane portions revealed almost no significant differences in bud break levels throughout the experiment when compared with the single nodes. This suggests that removal of the apical part of the cane would be effective in improving bud break by reducing the paradormant condition. A period of secondary dormancy was also observed in the intact canes which may also exacerbate the poor bud break observed in protected crops. This was not seen in the single nodes or the trisected canes which indicates that treatments which reduce paradormancy may also minimise the risk of secondary dormancy. By identifying the phase of bud dormancy which causes poor bud break, attention can now be focused on methods which overcome paradormancy in protected crops. Such methods might include tipping (removal of the cane apex), horizontal training methods, more efficient chilling methods, and chemical treatments.     

Incidence and genetic diversity of raspberry bushy dwarf virus (RBDV) in Rubus spp. in Turkey

Raspberry bushy dwarf virus (RBDV), recently renamed to Idaeovirus rubi, is one of the most common viruses infecting Rubus species worldwide but there is still a limited number of genome sequences available in the GenBank database and the majority of the sequences include partial sequences of RNA-1 and RNA-2. The distribution and incidence of RBDV in main raspberry and blackberry growing provinces in Turkey were monitored during 2015–2019 and 537 Rubus spp. samples were tested by both DAS-ELISA and RT-PCR. Among the tested samples, 36 samples tested positive for RBDV by DAS-ELISA and 67 samples by RT-PCR. There was relatively low nucleotide diversity among the Turkish isolates. Turkish isolates shared 93%–97.7%, 84.3%–98.9%, and 85%–99.2% nucleotide sequence identities with available sequences in the GenBank, in partial RNA-1, movement protein (MP) and coat protein (CP) genes, respectively. In the phylogenetic tree constructed for RNA-1, MP, and CP sequences, all Turkish raspberry isolates were clustered in a distinct clade. However, the blackberry isolates showed considerable variation in nucleotide sequences and were placed in three distinct groups. The divergent blackberry isolates showed high variability in MP (84.5%–89.3%) and CP (85.5%–89.7%) regions and were placed in a distinct group. The rest of blackberry isolates clustered together with sweet cherry RBDV isolates adjacent to the grapevine clade or together with raspberry isolates. The comparative analysis conducted on three RNA segments of RBDV highlighted the high sequence diversity of Turkish RBDV isolates. This study also emphasizes the importance of regular monitoring of RBDV infections in Turkey, with special regard to those Rubus spp. and grapevine accessions employed in conservation and selection programmes. In particular, the presence of new RBDV genetic variants and infection of Rubus species must be taken into account to choose a correct detection protocol and management strategy.     

Infectibility and sensitivity of UK raspberry, blackberry and hybrid berry cultivars to Rubus viruses

Six blackberry or hybrid berry cultivars and 19 raspberry cultivars were assessed for their infectibility with, and sensitivity to, graft inoculation with 10 distinct viruses found infecting Rubus in the UK. Cultivars were grafted with each of, two isolates of the pollen borne raspberry bushy dwarf virus (RBDV), five aphid borne viruses: black raspberry necrosis, raspberry leaf mottle (RLMV), raspberry leaf spot (RLSV), rubus yellow net and raspberry vein chlorosis (RVCV); and isolates of the nematode transmitted nepoviruses, arabis mosaic, raspberry ringspot, strawberry latent ringspot and tomato black ring. All tested cultivars were infectible with a resistance breaking isolate of RBDV but only about half of that number with the Scottish type isolate of the virus. The raspberry cvs Autumn Bliss, and occasionally Glen Garry and Glen Prosen, developed leaf yellowing symptoms following infection with RBDV, but none of the other infected cultivars showed obvious leaf symptoms when kept in a heated glasshouse during the growing season. All tested cultivars were infectible with each of the four viruses transmitted in nature by the aphid, Amphorophora idaei. Most were infected symptomlessly, but seven cultivars developed severe leaf spotting symptoms due to infection with RLMV or RLSV. All but one of the raspberry cultivars were infectible with RVCV, which is transmitted in nature by the aphid Aphis idaei, and almost all infected plants developed leaf symptoms; only one of the hybrid berry or blackberry cultivars tested was infected with RVCV. In tests with the four nepoviruses, all tested cultivars, except Tummelberry, were infectible with at least one or more of these viruses. However, cultivars responded differently to challenge inoculation with different isolates of individual nepoviruses. Several cultivars developed chlorotic leaf mottling following infection with some nepovirus isolates. The implications of these results for virus control are discussed in the light of the changing pattern of virus and virus vector incidence in the UK.     

Use of DNA fingerprinting for human population genetic studies

DNA fingerprinting techniques have been used in population genetic studies on many different kinds of organisms. Here, we present new applications for multilocus DNA fingerprint probes in population studies and demonstrate the applicability of DNA fingerprinting to human population genetics, using M13 phage DNA as a probe. The new approach, which is based on a factor method of numerical coding of non-quantitative data (factor correspondence analysis-FCA), shows good agreement between population position, as indicated by the three principal factors, and ethnogenetic proximity.     

Effectiveness of resistance genes to the large raspberry aphid, Amphorophora idaei Börner, in different raspberry (Rubus idaeus L.) genotypes and under different environmental conditions

The introduction into commerce of raspberry cultivars with major gene resistance to the large raspberry aphid, Amphorophora idaei, an important pest and virus vector on red raspberry in Europe, has been very effective both in decreasing pest numbers and greatly restricting infection with the viruses it transmits. However, biotypes of the aphid able to overcome these genes have developed in the field in recent years. Additionally, in field and laboratory tests, the response to aphid biotypes and recognised aphid strains of certain raspberry cultivars, such as Glen Prosen and Delight, differ markedly despite the fact that they are reputed to contain the same A. idaei‐resistance gene, A₁. In attempts to understand the reasons for this difference in response, analysis was made of the segregation of progeny seedlings from crosses between A. idaei‐resistant and ‐susceptible cultivars to two recognised strains of the aphid. These studies showed that, as expected, cv. Autumn Bliss contained the A. idaei‐resistance gene, A₁₀, and cvs Delight and Glen Prosen each contained the A. idaei‐resistance gene, A₁. When progeny seedlings were assayed in a heated glasshouse as young plants and in an unheated Tygan house as 1 m tall plants, the segregation ratios for resistance and susceptibility to A. idaei were largely unchanged. However, when the resistance of individual progeny plants was assessed, c. 37% of the putative gene A₁‐containing progeny and 9–23% of the putative gene A₁₀‐containing progeny, behaved differently in these two environments. Experiments involving an A. idaei‐resistant and ‐susceptible parent cultivar showed that shading plants increased their susceptibility to A. idaei colonisation. Whilst this shading effect has implications for experimentally detecting A. idaei‐resistant progeny in segregating raspberry seedlings, it does not explain the difference in field resistance to A. idaei of cvs Delight and Glen Prosen. Such differences in the field seem best explained by the presence in these cultivars of ‘minor’ genes for A. idaei resistance and/or susceptibility that influences the effectiveness of gene A₁.     

Peg-mediated fusion of Rubus idaeus (raspberry) and R. fruticosus (blackberry) protoplasts,selection and characterisation of callus lines

ABSTRACT

Cell suspension-derived protoplasts of two cultivated Rubus species, Rubus idaeus-raspberry (subgenus Idaeobatus 2n=2x=14) and R. fruticosus-blackberry (a complex species aggregate within the subgenus Eubatus, 2n=4x=28) were fused using different polyethylene glycol (PEG) fusion treatments. Duration of PEG treatment and choice of culture media influenced the rate of cell divisions and plating efficiency. Colony formation was initiated on solid media for the production of several callus lines. Cytological analyses were performed on selected callus lines with hexaploid chromosome number. Two hexaploid fusion callus lines, selected for their homogeneity in growth and ploidy level, were examined by molecular cytogenetic techniques of fluorescent in situ hybridisation (FISH) and genomic in situ hybridisation (GISH). GISH revealed the presence of the heterokaryon within the fusion callus lines. FISH probed with ribosomal DNA (rDNA) showed variable numbers and sizes of loci. Aberrant distribution and condensation of rDNA were common in interphase cells. FISH results suggest that large karyotype rearrangements occurred, including variation in chromosome number and rDNA loci translocations. Attempts to regenerate plants from the hexaploid callus lines following several applications of plant growth regulator combinations were unsuccessful. This may be attributed to the genomic reorganisation and instability of these long-term fusion callus cultures.     

Genotypic diversity revealed by allozymes and oligonucleotide DNA fingerprinting in French populations of the aquatic macrophyte Sparganium erectum

The vast majority of perennial plants reproduce sexually and vegetatively at the same time. This may lead to important variation among clonal plant populations in their amount of genotypic diversity. In order to verify this assumption, we compare the clonal diversity of 10 natural populations of the aquatic clonal macrophyte Sparganium erectum in France. Diversity was quantified by DNA fingerprinting and allozyme electrophoresis for a sample of 10 shoots per population. Two DNA probes (CA)₈ and (TAA)₆ TA, were selected among 10 synthetic oligonucleotide probes containing simple repeat motifs. Both allozymes and DNA fingerprints revealed different amounts of diversity among populations. Five populations consist of a single genotype, whereas two populations were genetically highly diverse. In four of the monomorphic populations, absence of fingerprints diversity was combined with uniformly heterozygous allozyme loci, suggesting that each population was composed of a single clone. In the highly diverse populations, the level of clonal diversity combined with the allele segregation of the two allozyme loci Lap and Est suggests frequent seedling recruitment. The origin of new genotypes remains unclear but the absence of widespread genotypes together with the discrete distribution of Sparganium erectum populations implies that new genotypes are locally produced through sexual reproduction.     

Genetic variation of the St. Lawrence beluga whale population assessed by DNA fingerprinting

Recent surveys suggest that the endangered St. Lawrence beluga ( Delphinapterus leucas ) population is not recovering significantly despite 20 years of protection. Dead individuals that have been autopsied show high levels of tumours and infections. This situation could be a result of pollution, loss of genetic variation, inbreeding depression or a combination of these factors. Analyses of DNA fingerprints from St. Lawrence belugas with three minisatellite probes (Jeffreys 33.6, 33.15 and M13) indicate a reduced level of genetic variation compared to Beaufort Sea animals. The average band-sharing between individuals of the St. Lawrence beluga population for the three probes (0.534, 0.573 and 0.478, respectively) was significantly higher than that of the Beaufort Sea beluga population (0.343, 0.424, 0.314, respectively). Higher levels of mean allele frequency in the St. Lawrence belugas (0.33 vs. 0.21) suggest that this population is composed of individuals which are related. Inbreeding depression could therefore be a factor in the lack of recovery of the St. Lawrence beluga population.     

Increase of genetic variation over time in a recently founded population of great reed warblers (Acrocephalus arundinaceus) revealed by microsatellites and DNA fingerprinting

Genetic similarity within pairs of individuals was examined using both 10 polymorphic microsatellite loci and multi-locus DNA fingerprinting profiles in a semi-isolated population of great reed warblers at Lake Kvismaren, south Central Sweden, in 1987-1993. The population was founded by a few individuals in 1978, followed by a gradual increase in numbers until 1988, since when the population has remained relatively stable with about 60 breeding birds. We have previously found that high genetic similarity between pair-mates in the population during the early part of the study period reduced egg hatching success, and hence reproductive success. The measures of pairwise genetic similarity, microsatellite allele sharing and DNA fingerprinting band sharing, were highly correlated with pedigree-based relatedness. Both microsatellite and DNA fingerprinting similarities between pair-mates declined significantly over the study period, and the pattern was most pronounced in the DNA fingerprinting data. Analyses restricted to the microsatellite data showed that the average annual microsatellite similarity between pairwise combinations of individuals, as well as individual homozygosity in males, declined significantly over the study period, and that several immigrants carrying novel alleles entered the population during the study. Hence, the temporal decline in genetic similarity of mates in the population is probably a consequence of increased immigration, facilitated by the recent expansion of the species in the region. These results suggest that the population has now recovered genetically, or is in the process of recovering, from a recent founder event.     

Monogamy in black vultures: genetic evidence from DNA fingerprinting

Recent research has indicated that avian mating systems maycommonly deviate from monogamy due to extrapair fertilizations(EPFs). Because the majority of avian species have long beenconsidered monogamous, accurate measurement of the frequencyof EPFs in a variety of species is important to enhance understandingof the evolution of avian mating systems. We used DNA fingerprintingto investigate the apparently monogamous mating system of blackvultures (Coragyps airaius) by assaying parentage within severalnuclear families. Monogamy is suggested in black vultures becausemated pairs exhibit long-term pair bonding and year-round association,and share incubation and nestling feeding duties equally. Thirtytwobreeders and 36 nestlings representing 16 complete nuclear familieswere tagged for individual identification and sampled for DNAanalysis using 2 restriction enzymes and 3 probes for hypervariableregions. Putative parents were assigned parentage in all cases.We empirically examined the probability of detecting EPFs bycomparing nestlings' fingerprints to those of a putative parentand another randomly chosen adult. All putative parents couldbe assigned with 95%confidence and all outside adults couldbe similarly excluded. There is therefore no evidence for successfulEPFs in this population, indicating a mating system that doesnot deviate from strict monogamy. The complex social behaviorof black vultures may eliminate the opportunity for EPFs dueto the prohibition of copulations in the presence of relatives.     

Assessment of DNA fingerprinting for determining genetic diversity in sheep populations

Genomic DNA, prepared from 12 animals from four sheep flocks, was digested with either HaeIII or Hin fI and probed with three DNA fingerprinting probes. Mean DNA fingerprint band sharing and band frequency calculated for each flock were used to estimate genetic diversity. Each of the DNA fingerprinting systems showed the same trend in diversity within the sampled flocks, and greater diversity between the flocks than within the flocks. DNA fingerprinting therefore provides a useful measure of genetic diversity in sheep.     

DNA fingerprinting: a tool for determining genetic distances between strains of poultry

Summary DNA fingerprinting, a technique based on the detection of hypervariable minisatellite regions in DNA restriction fragments, was tested for its applicability to conduct population genetics in poultry. Using MspI digestion and phage M13 DNA as a probe, between 25 and 35 minisatellite-containing DNA fragments were observed per bird. Comparison of the banding pattern of offspring with their parents revealed that the bands were inherited as stable genetic traits. The variability of the DNA fingerprinting pattern was reduced in inbred strains. DNA fingerprints of chickens from five well-defined populations of known genetic relationships were analyzed and indices of genetic distances were computed. They correctly reflected the history of these strains, indicating that DNA fingerprinting may be a powerful tool to characterize genetic relationships between different breeding populations of the same species.     

Introduced Swedish Canada geese (Branta canadensis) have low levels of genetic variation as revealed by DNA fingerprinting

The Swedish, Finnish and Norwegian population of Canada geese (Branta canadensis), now amounting to some 30–50 000 birds, was founded by only five individuals. We used DNA fingerprinting to assess the level of genetic variability in minisatellite loci of Swedish Canada geese from two northern areas. For comparison, we estimated the minisatellite variability in lesser white-fronted geese (Anser erythropus), barnacle geese (Branta leucopsis) and a reintroduced stock of Canadian giant Canada geese (Branta canadensis maxima). The mean similarity between Swedish Canada geese was 0.76 ± 0.15, which is higher than recorded for any other natural bird population. The high similarity implies that a fourfold increase of homozygosity has taken place in this population. The probable cause for the loss of variation is the low number of birds originally introduced and a history of repeated translocations, leading to a sequence of founder events. As a consequence of the high similarity, it has not been possible to use DNA fingerprinting for determination of parenthood in the population studied.     

DNA fingerprinting in three species of neotropical primates

DNA fingerprinting allows the simultaneous detection of a large number of hypervariable loci consisting of highly polymorphic tandem repeat units that are extensively dispersed in the genome. With the 33.6 human minisatellite probe, hypervariable fragments were detected, for the first time, in the genome of three different species of wild-caught neotropical primates: Aotus infulatus, Aotus azarae, and Cebus apella. As in the human, these species were highly polymorphic, showing distinctive, individual-specific patterns. Estimates of relatedness within each group were calculated from interspecific comparisons based on the number of shared fragments between individuals. This work shows that the 33.6 human minisatellite probe can be very useful for increasing our understanding of population dynamics and behavior of these species in their natural habitat. © 1996 Wiley-Liss, Inc.     

DNA fingerprint variation in some blackberry species (Rubus subg.Rubus,Rosaceae)

We have analysed samples from Sweden, Denmark, and Germany of six facultatively apomictic blackberry species to investigate the accordance between a taxonomy based on morphological characters on the one hand, and distribution of genetic variation estimated by DNA fingerprinting on the other hand. DNA fingerprint variation was found to be quite restricted in all species investigated. The first taxonomic group included three species related toRubus nessensis, two being characterized by one very widespread DNA fingerprint in all three countries and a few rare ones, whereas the third species differed between Sweden and Germany. The second taxonomic group included species related toR. gracilis. Two of these species exhibited very similar DNA fingerprints, whereas the third species deviated clearly. The utilization of DNA fingerprinting as a tool in taxonomy is discussed; most likely this method could become a useful complement to morphology, especially in plant groups with reduced levels of genetic recombination.     

DNA fingerprinting in horses using a simple (TG)n probe and its application to population comparisons

A synthetic polynucleotide (TG)_n was hybridized to equine DNA digested with HinfI and hypervariable hybridization patterns were obtained. Mendelian inheritance of these DNA fingerprinting patterns was confirmed by pedigree analysis. Estimates of the probabilities of identical band patterns in unrelated individuals of different breeds (Swedish Trotters, North Swedish Trotters, Thoroughbreds and Arabians) were in the range 1 times 10-⁴-7 times 10-⁶ The variability derived with the (TG)_n, probe in horses was higher than what we obtained with several other commmonly used probes for DNA fingerprinting. Individuals within breeds tended to be more similar to each other with regard to DNA fingerprint pattern than to individuals of other breeds. Moreover, a parsimony analysis made on the basis of the hybridization patterns gave clustering of individuals within breeds. The possibility of using hypervariable probes for the identification of breed-specific characters is discussed.     

Detection of leucochimaerism in bovine twins by DNA fingerprinting

Karyotyping and hypervariable genetic markers indicate extensive leucochimaerism between pairs of dizygotic twins in cattle, a result of placental vascular anastomosis. The extent of this chimaerism includes both kind and number of cells exchanged. All heterosexual twin pairs harboured two types of leucocytes, having either XX or XY chromosome pairs, and 30 of 31 pairs of twins shared identical DNA fingerprints. Although chromosome results from skin fibroblasts indicate that some chimaerism occurs in the skin, the low level allows for differentiation of genotypes between twins. The results warrant against the common practice of using blood samples for DNA typing if twinning is not properly documented.