Microsatellite repeats in grapevine reveal DNA polymorphisms when analysed as sequence-tagged sites (STSs)

Microsatellite repeat sequences were investigated as sequenced-tagged site (STS) DNA markers to determine the potential for genetic analysis of the grapevine genome. The PCR-generated markers detect codominant alleles at a single locus or site in the genome. The marker type is very informative detecting high heterozygosity (69%–88%) within individual grapevine cultivars and high genetic variation between cultivars, making it a useful marker type for plant genome mapping and genome typing. For five loci a screening of 26 V. vinifera cultivars found 13, 12, 8, 5, and 4 different length alleles respectively with some alleles more common than others. The genomic DNA sequences surrounding microsatellite sequences were conserved within the genus permitting STS primers to amplify STSs from other Vitis species. These Vitis species were found to have some unique alleles not present in V. vinifera.     

Polymorphisms in grapevine DNA detected by the RAPD PCR technique

A sensitivie, reproducible technique is described for detecting polymorphisms between the nuclear DNA of grapevine isolates using the RAPD PCR technique. Unique fingerprints of a number of cultivars were readily distinguished by using either single primers or mixtures of two primers. The method will be used to provide a databank of fingerprints for the rapid identification of grapevine cultivars, and to develop phylogenetic relationships. It will also be extended and modified in an attempt to detect polymorphisms between DNAs of clonal selections of individual cultivars.     

A simple and efficient method for DNA extraction from grapevine cultivars andVitis species

A quick, simple, and reliable method for the extraction of DNA from grapevine species, hybrids, andAmpelopsis brevipedunculata (Vitaceae) has been developed. This method, based on that of Doyle and Doyle (1990), is a CTBA-based extraction procedure modified by the use of NaCl to remove polysaccharides and PVP to eliminate polyphenols during DNA purification. The method has also been used successfully for extraction of total DNA from other fruit species such as apple (Malus domestica), apricot (Prunus armeniaca), cherry (Prunus avium), peach (Prunus persica), plum (Prunus domestica), and raspberry (Rubus idaeus). DNA yield from this procedure is high (up to 1 mg/g of leaf tissue). DNA is completely digestible with restriction endonucleases and amplifiable in the polymerase chain reaction (PCR), indicating freedom from common contaminating compounds.     

Using randomly amplified polymorphic DNA for evaluating genetic relationships among papaya cultivars

Summary We have applied the recently developed technique of random amplification of polymorphic DNA (RAPD) to the analysis of the relationships among ten cultivars of papaya (Carica papaya L.). Eleven ten-base synthetic oligonucleotides were chosen that gave multiple PCR amplification products using papaya DNA as template. These 11 primers amplified a total of 102 distinct fragments. Cultivars were scored for presence or absence of RAPD fragments and grouped by cluster analysis using simple matching coefficients of similarity. A dendrogram of the ten cultivars was constructed. Of the ten cultivars seven were of the Hawaiian type, and all of these grouped to one branch of the tree. Divisions within the Hawaiian, branch were mostly consistent with the known genetic background of these cultivars. Three non-Hawaiian, cultivars were also analyzed. The minimum similarity detected was 0.7 suggesting that the domesticated papaya germ plasm is quite narrow. Our results show that RAPD technology is a rapid, precise and sensitive technique for genomic analysis.Journal series No. 3692 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification

Summary Repetitive DNA sequences present in the grapevine genome were investigated as probes for distinguishing species and cultivars. Microsatellite sequences, minisatellite sequences, tandemly arrayed genes and highly repetitive grapevine sequences were studied. The relative abundance of microsatellite and minisatellite DNA in the genome varied with the repeat sequence and determined their usefulness in detecting RFLPs. Cloned Vitis ribosomal repeat units were characterised and showed length heterogeneity (9.14–12.15 kb) between and within species. A highly repetitive DNA sequence isolated from V. vinifera was found to be specific only to those species classified as Euvitis. DNA polymorphisms were found between Vitis species and between cultivars of V. vinifera with all classes of repeat DNA sequences studied. DNA sequences suitable for DNA fingerprinting gave genotype-specific patterns for all of the cultivars and species examined. The DNA polymorphisms detected indicates a moderate to high level of heterozygosity in grapevine cultivars.On leave from the Biochemical Research Institute, Nippon Menard Cosmetic Co, Ltd, Ogaki Gifuken, 503 Japan     

Hypervariable single and multi-locus DNA polymorphisms for genetic typing of non-human primates

The series of hypervariable, “minisatellite” loci characterized byJeffreys and coworkers in the human myoglobin gene have proved to be DNA sequences highly conserved throughout the eukaryotic genome, and hence the methodology developed for human DNA “fingerprinting” has found immediate application in an ever expanding number of species. Primatologists have not been slow to profit from a method which predicts individual recognition to a very high degree of probability, and initial studies have focused on paternity allocation (rather than paternity exclusion, as designated by the classical biochemical markers), adaptive aspects of socio-sexual behaviour patterns and mating systems. A number of probes with sequences corresponding to the common minisatellite core sequences have been used for probing genomic DNA, and synthetic, G-rich oligonucleotides (15 – 37 bases), corresponding to the core sequence of the minisatellite repeat unit, or simply di-, tri-, or tetranucleotide repeats, appear to be equally discriminatory. The multiple banding patterns produced on hybridization of these probes to restriction enzyme digests of DNA provide an advantage in that the probability of two unrelated individuals sharing the same banding pattern will be low. However, the uncertainty of linkage of the multiple loci identified precludes genotyping and population genetic analyses based on allele frequencies. In contrast, single locus analysis allows DNA typing using variable number tandem repeat (VNTR) or restriction fragment length (RFLP) DNA polymorphisms, and the merits and drawbacks relative to DNA fingerprinting are discussed. For the behavioural primatologists dealing with defined, accessible troops of primates, the value of multilocus DNA fingerprinting, in terms of established methodology and availability of probes applicable to species as phylogenetically wide-ranging as apes and prosimians, may well outweigh the loss of genotypic and population structure data.     

Experimental verification of microsatellite null alleles in Norway spruce (Picea abies [L.] Karst.): Implications for population genetic studies

Three stands ofPicea abies [L.] Karst. with different density in the Harz Mountains (Lower Saxony, Germany) were characterized at 4 microsatellite loci. An excess of homozygotes was observed in all 3 stands at 1 simple sequence repeat (SSR) locus, suggesting the presence of null alleles. To test for the segregation of a null allele, 24 openpollinated seeds (haploid megagametophytes and embryos) from apparently homozygous mother trees were analyzed. For 1 of 3 trees that could be identified as heterozygous for a null allele, no significant deviation from the expected 1∶1 segregation into marker absence (null allele) and marker presence of the second maternal allele could be observed in the haploid megagametophyte. Concordantly, the numbers of embryos heterozygous for the null allele and for the other maternal allele were not significantly different from each other. Inheritance analyses in seedlings and corresponding megagametophytes of gymnosperms were used as a direct experimental verification of microsatellite null alleles in single-tree progeny. Microsatellites with an abundance of null alleles should be discarded from further analysis because inclusion of these loci results in incorrect estimation of allele frequencies.     

Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets

This study focused on haploid induction in mandarin through in situ gynogenesis by pollination with irradiated pollen of ‘Meyer’ lemon. Pollination was carried out for three genotypes of mandarin with four levels of gamma-ray-irradiated pollen (150, 300, 600, and 900 Gy). The resulting seeds were characterised by a small size. Embryos were rescued in vitro and the ploidy level of the plantlets was determined by flow cytometry analysis. Haploid, diploid, triploid plantlets were obtained. The haploid parthenogenetic origin was confirmed using microsatellite marker analysis and chromosome count. Diploid and triploid plants were the result of crosses between mandarin and lemon. The induction of gynogenetic haploids of ‘Fortune’ (Citrus clementina Hort ex Tan. × Citrus tangerina Hort ex Tan.) and ‘Ellendale’ (Citrus reticulata Blanco × Citrus sinensis L. Osb) is reported here for the first time.     

Inheritance of the human salivary proline-rich proteins: A reinterpretation in terms of six loci forming two subfamilies

DNA studies suggest that six loci control the synthesis of human salivary proline-rich proteins (PRPs). Genes at two of these loci (proposed names, PRH1 and PRH2) contain regions that strongly hybridize to a probe made from a cDNA in which sites for the restriction enzyme HaeIII occur repeatedly; they code for the acidic PRPs. Genes at the remaining four loci (PRB1, PRB2, PRB3, and PRB4) contain regions that strongly hybridize to a probe with repeated BstN1 sites; they probably code for the basic and glycosylated PRPs. In contrast to these data suggesting six loci forming two gene subfamilies, studies of protein polymorphisms and families have led to the postulation of 13 loci with 11 common null alleles. The discrepancy in the number of loci is partly resolved by the hypothesis that the three acidic PRPs, Db, Pa, and PIF, are coded for by alleles at one of the HaeIII-type loci rather than by three discrete loci.This work was supported by National Institutes of Health Grants DEO 3658-19 (E.A.) and GM 20069 (O.S.). This is paper No. 2774 from the Laboratory of Genetics, University of Wisconsin—Madison.     

Genetic relatedness in the antCamponotus herculeanus. A comparison of estimates from allozyme and DNA microsatellite markers

Summary Genetic structure ofCamponotus herculeanus populations was studied using both traditional allozyme markers and recently discovered DNA microsatellites. Relatedness among worker nest mates was high in one study population (0.70), but lower in another (0.48). The estimates lower than 0.75, together with the multilocus genotype patterns, indicate either multiple mating by queens, or multiple queens reproducing in the colonies. These two could not be distinguished with the current data, although multiple matings seem a more probable explanation for the relatedness estimates bearing social biology of the species in mind. Inbreeding coefficients suggested random mating in both populations. Both allozyme and DNA microsatellite markers gave the same result concerning genetic parameters estimated, but the higher variability of the microsatellites resulted in more precise estimates of both parameters.     

An improved procedure for the isolation of nuclear DNA from leaves of wild grapevine dried with silica gel

An efficient and convenient method is presented for the isolation of nuclear DNA from leaves of wildVitis species that have been dried with silica gel. The nuclear DNA obtained with this method is suitable for both PCR amplification and digestion with restriction endonucleases.     

Optimization of primer screening for evaluation of genetic relationship in rose cultivars

Optimization of primer screening for evaluation of genetic relationship in 34 cultivars of rose through random amplified polymorphic DNA (RAPD) markers was investigated. Four series of decamer primers were used for screening and optimization of RAPD analysis between which A and N series performed good amplification of fragments as compared with other series. The primers OPN-07 and OPN-15 produced maximum number of DNA fragments in Rosa hybrida cv. Anuraag. Some primer either did not produce amplification or produced very poor amplification. Further, ten selected primers were used for genetic analysis of 34 rose cultivars. The primer OPN-15 amplified 21 fragments in all cultivars tested. A total of 162 distinct DNA fragments (bands) ranging from 100 to 3400 base pairs were amplified by using 10 selected random primers. The cluster analysis indicated that these rose cultivars formed nine clusters.     

Prospectives for applying molecular and genetic methodology to improve wheat cultivars in drought environments

With the advent of molecular biotechnologies, new opportunities are available for plant physiologists to study the relationships between wheat traits and their genetic control. The functional determinations of all genes that participate in drought adaptation or tolerance reactions are expected to provide an integrated understanding of the biochemical and physiological basis of stress responses in wheat. However, despite all the recent technological breakthroughs, the overall contribution of genomics-assisted breeding to the release of drought-resilient wheat cultivars has so far been marginal. This paper critically analyses how biotechnological, genetic and information tools can contribute to accelerating the release of improved, drought-tolerant wheat cultivars. Armed with such information from established models, it will be possible to elucidate the physiological basis of drought tolerance and to select genotypes with an improved yield under water-limited conditions.     

Rapid estimation of genetic relatedness among heterogeneous populations of alfalfa by random amplification of bulked genomic DNA samples

A procedure which involves the use of RAPD markers, obtained from bulked genomic DNA samples, to estimate genetic relatedness among heterogeneous populations is demonstrated in this study. Bulked samples of genomic DNA from several alfalfa plants per population were used as templates in polymerase chain reactions with different random primers to produce RAPD patterns. The results show that the RAPD patterns can be used to determine genetic distances among heterogeneous populations and cultivars which correspond to their known relatedness. The results also indicate that, by using ten primers with bulked DNA samples from ten individuals, 18–72 populations or cultivars can be distinguished from each other on the basis of at least one unique RAPD marker. We anticipate that DNA bulking and methods for comparing RAPD patterns will be very useful for identifying cultivars, for studying phylogenetic relationships among heterogeneous populations and for selecting parents to maximize heterosis in crosses.     

Individualization and estimation of relatedness in crocodilians by DNA fingerprinting with a Bkm-derived probe

Individual-specific DNA fingerprints of crocodilians were obtained by the use of Bkm-2(8) probe. Pedigree analyses of Crocodylus palustris, C. porosus and Caiman crocodilus revealed that the multiple bands (22–23 bands with Aludigest) thus obtained were inherited stably in a Mendelian fashion. Unique fingerprints permitted us to identify individuals, assign parentage, and reconstruct the DNA profile of a missing parent. Average band sharing between unrelated crocodiles was found to be 0.37. Band sharing between animals of known pedigrees increased predictably with relatedness and provided a basis for distinguishing relatives from non-relatives. Similar results obtained in other species/genera, using the same probe, suggest that this approach may be applicable to all species of crocodilians, and could facilitate genetic studies of wild and captive populations.     

Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P≤0.05) were detected between diploid and parental field plants. No haploid or aneuploid plants were observed. The nuclear DNA content of nine V. vinifera cultivars was also estimated using flow cytometry. A non-significant variation was found among the cultivars, with DNA content ranging from 1.17 pg/2C (cv. ‘Tinta Barroca’ and ‘Viosinho’) to 1.26 pg/2C (cv. ‘Cabernet Sauvignon’). These results and previous studies on other Vitis species suggest that Vitis genome is stable with regard to nuclear DNA content.     

Cloacal and buccal swabs are a reliable source of DNA for microsatellite genotyping of reptiles

In this study, a minimally invasive method for DNA sampling of reptiles and amphibians using cloacal and buccal swabs is described. High molecular weight DNA was isolated from the swabs, which were collected from tuatara (Sphenodon punctatus), and stored in 70% ethanol at room temperature for approximately 1 week. Amplification of mitochondrial and microsatellite DNA loci was successful from both cloacal and buccal swabs, and in all cases the genotypes matched those obtained from blood samples. These results show that cloacal and/or buccal swabbing is a useful alternative to blood sampling and toe clipping for genetic studies on reptiles. This method is rapid, inexpensive and easy to implement in field situations.     

Structural organization and differential expression of three stilbene synthase genes located on a 13 kb grapevine DNA fragment

A 13 kb DNA fragment was isolated from a grapevine (Vitis var. Optima) genomic library by hybridizing with elicitor-induced stilbene synthase cDNA as a probe. After fragmentation with Eco RI, subcloning and sequencing, two full-size stilbene synthase genes (Vst1 and Vst2) and the 3 end of a third stilbene synthase gene (Vst3) were located within the 13 kb fragment. Vst1 and Vst2, differing only slightly in the coding region, are distinguished in the intron size and in the structure of the promoter region. The 5 flanking region of gene Vst1 contains a TATAA box at nucleotide –48. The substantial structural differences found for the promoters of the two genes are paralleled by a striking difference in the expression of the two genes in elicitor-treated cells. Moreover, the accumulation upon elicitation of six different stilbene synthase mRNAs was studied and found to differ by two orders of magnitude.     

Diversity of DNA methylation pattern and total DNA restriction pattern in symbiotic Nostoc

Attempts were made to use total DNA restriction patterns and the response of purified DNA to treatment with restriction endonucleases to characterize several symbiotic Nostoc strains which had been isolated from different host plants cultivated in Italy. Among 27 restriction endonucleases tested, several did not cut any DNA and no significant variation in the susceptibility of the genomes to DNA restriction was seen among the strains. Therefore the Nostoc strains could not be separated into groups based on their different susceptibilities to the action of restriction endonucleases. However, in studies of total DNA restriction patterns, the restriction endonucleases BfrI and HpaI gave unique band patterns for each cyanobacterial isolate. Different profiles were even found in strains isolated from host plants belonging to the same species. The results do not support any definition of symbiotic Nostoc genomic groups or species and show that a tight specificity between the host plant and the cyanobacterium might not exist in the symbiotic associations involving Nostoc.