Microsatellite marker-based identification and genetic relationships of olive cultivars from the Extremadura region of Spain

Seventy-seven olive accessions corresponding to 25 cultivars from the Extremadura region of Spain were studied using four microsatellite or SSR markers in order to fingerprint them, and evaluate genetic similarity and relationships between local and introduced olive cultivars. The number of alleles per locus ranged from 4 to 8, with a mean of 6.25 alleles per primer pair (a total of 25 alleles). The observed heterozygosity ranged from 0.58 to 0.95, while the expected heterozygosity varied between 0.68 and 0.83. The polymorphism information content values ranged from 0.63 to 0.79. The mean polymorphism information content value of 0.70 for the SSR loci provided sufficient discriminating ability to evaluate the genetic diversity among the cultivars. The SSR data allowed unequivocal identification of all the cultivars; a combination of three SSR markers was sufficient to discriminate all 25 olive cultivars. A dendrogram was prepared, using the unweighted pair-group method with arithmetic mean clustering algorithm; it depicted the pattern of relationships between the cultivars. Most of the local cultivars grouped according to their geographic origin. No clear clustering trends were observed when the morphological traits of fruit endocarps or fruit use of cultivars were employed as analysis criteria. We conclude that there is a high level of variability among local olive cultivars from the Extremadura region at both the morphological and molecular levels; these data should be useful for identifying and distinguishing local germplasm.     

A new strategy employed for identification of sweet orange cultivars with RAPD markers

We optimized RAPD techniques by increasing the length of RAPD primers and performing a strict screening of PCR annealing temperature to distinguish 60 sweet orange cultivars from the Research Institute of Pomology at the Chinese Academy of Agricultural Sciences. A new approach called cultivar identification diagram (CID) was used to improve the efficiency of RAPD markers for cultivar identification. Thirteen effective primers were first screened from 54 RAPD arbitrary 11-mer primers based on their amplification products and amplified polymorphic bands; they were then used for PCR amplification of all 60 cultivars. All cultivars were manually and completely separated by the polymorphic bands appearing in DNA fingerprints from 13 primers; a CID of the 60 sweet orange cultivars was then constructed. This CID separated all the cultivars from each other, based on the polymorphic bands; the corresponding primers were marked in the correct positions on the sweet orange CID. The CID strategy facilitates the identification of fruit cultivars with DNA markers. This CID of sweet orange cultivars will be very useful for the protection of cultivar rights and for early identification of seedlings in the nursery industry.     

Partial molecular characterization of some kiwi fruit cultivars by RAPD markers

Molecular variability among seven cultivars of A. deliciosa var. deliciosa was investigated through RAPD markers. Thirty four decamer primers were screened generating polymorphic patterns of amplified DNA for these cultivars. Twenty one selected primers gave clear and reporducible patterns. A total of 430 bands were produced and 29.37% of them were polymorphic. The patterns distinguished between the cultivars and their analysis established an approach to classification within A. deliciosa var. deliciosa based on RAPD markers. The dendrogram clearly differentiated male from female cultivars. While abbot and allison female cultivars were closely related, bruno and abbot female cultivars showed maximum dissimilarity.     

A novel strategy for identification of 47 pomegranate (Punica granatum) cultivars using RAPD markers

DNA marker can be used for precise plant cultivar identification. However, DNA markers have often not been used effectively for the identification of plant cultivars due to a lack of an effective analysis strategy. We used a novel strategy for effective identification of plant individuals based on a new way of recording DNA fingerprints of the genotyped plants; a cultivar identification diagram can be manually generated and used as key reference information for quick identification of plant and/or seed samples. Forty-seven pomegranate varieties popularly cultivated in various provinces of China were subjected to RAPD marker analysis. Using the cultivar identification diagram strategy, they were clearly separated by the fingerprints of 11 RAPD primers. The utility and accuracy of the cultivar identification diagram analysis results were confirmed by the identification of three randomly chosen groups of cultivars among the 47 varieties.     

Identification and classification of celery cultivars with RAPD markers

Summary Twenty-one celery (Apium graveolens L. var. dulce) cultivars, one celeriac (var. rapaceum) and one annual smallage (var. secalinum) cultivar were screened for polymorphic RAPD (Random Amplified Polymorphic DNA) markers with 28 arbitrary 10-mer primers. Among a total of 309 bands observed, 29 (9.3%) were polymorphic in the 23 cultivars screened, but only 19 (6.1%) markers were polymorphic within the 21 type dulce cultivars. These markers were sufficient to distinguish each of the cultivars used. The average marker difference was 6.4 between two celery cultivars, 16.7 between celery and annual smallage, 14.7 between celery and celeriac, and 12.0 between annual smallage and celeriac. The celery cultivars surveyed were classified into three groups based on the marker differences. The relationship among the dulce-type cultivars concluded from this research is basically consistent with the known lineage of the cultivars and the previous study using stem protein and isozyme markers. RAPD technology provides a new alternative for cultivar identification and classification in celery.     

RAPD markers for the identification of yield traits in tomatoes under heat stress via bulked segregant analysis

Tomato production is limited to a large extent by climates with high temperatures. Yield-related traits in tomatoes are generally thought to be quantitative inherited traits that are significantly affected by variation in environmental factors. Breeding for heat tolerance is restricted due to the complexity of the traits. The objective of this study was to identify random amplified polymorphic DNA (RAPD) markers linked to heat-tolerance traits in tomatoes under heat stress. Forty-three F(7) recombinant inbred lines (RILs) derived from a wild cross between CL 5915 (heat-tolerant) and L4422 (heat-sensitive) were obtained and scored for flower number, fruit number, fruit set, fruit weight and yield under screen house conditions during the hot (summer) season of 2003. The distributions of average fruit weight, fruit number, fruit set and yield in the F(7) population were strongly skewed towards heat susceptibility, characteristic of L4422. Significant positive correlation was observed between fruit weight and yield, and between fruit number, fruit set and yield. However, the increase in yield and fruit set by selecting for large flower number may be fairly minor due to non-significant correlation between these traits. Selecting for flower number may not be a useful tool for improving yield under heat stress. A total of 200 RAPD primers were screened, among which 14 were identified as associated with heat-tolerance using bulk segregant analysis (BSA) based on the F(7) population grown in a heat-stressed environment. Some RAPD markers were unique to one specific trait, and others were linked to two traits. All markers for heat tolerance related traits had positive gene effects as a result of the contribution of the CL5915 gene that bolstered these traits. One hundred F(2) plants derived from the same parents (CL5915xL4422) were grown in the same location during the summer of 2004 to test for the stability and reliability of the 14 markers identified. Selection for the desired heat-tolerance genotypes corresponded well with targeting heat tolerance traits using the selected heat tolerance RAPD markers identified. Marker-assisted selection (MAS) for heat tolerance may be efficiently conducted by selecting individuals that contain high fruit number, high fruit weight, and high yield markers (P06, X01, D06 and D11), which would thus facilitate conventional breeding using CL5915 as a donor parent.     

Molecular characterization of olive (Olea europaea L.) Sicilian cultivars using SSR markers

Olive (Olea europaea L.) is one of the economically most important fruit crops for the Mediterranean area, with production being mainly destined to oil extraction. In Sicily, olive has been cultivated since ancient times and its germplasm is characterized by a wide genetic diversity that could be related to its domestication and spread in ancient times, and to some reproductive biological peculiarities as self-incompatibility. This analysis was conducted on 65 genotypes with the purpose of characterizing a large collection of Sicilian accessions (47 genotypes) and to compare them with varieties coming from Southern Italy and from the most important countries of the Mediterranean basin. With this aim we used 8 simple sequence repeat (SSR) markers, which detected a total of 74 alleles and identified an average of 19.5 genotypes in the population investigated. A larger variability than expected was found in the analyzed genotypes, some synonymies already reported in literature were confirmed, but also some cultivars considered as identical were discriminated such as in the case of Castriciana, Ogliarola messinese and Passalunara. The whole study revealed a wide intraspecific variability within the gene pool examined, independently from the geographical origin.     

Identification of broccoli and cauliflower cultivars with RAPD markers

Summary RAPD (Random Amplified Polymorphic DNA) markers generated by 4 arbitrary 10-mer primers, discriminated 14 broccoli and 12 cauliflower cultivars (Brassica oleracea L.) by banding profiles. The size of the amplified DNA fragments ranged from 300 to 2600 base pairs. Twenty-eight percent of the markers were fixed in both broccoli and cauliflower, whereas 12.5% were specific to either crop. The rest were polymorphic in either or both crops. The markers generated by two and three primers were sufficient to distinguish each of the broccoli and cauliflower cultivars, respectively. The average difference in markers was 14.5 between broccoli and cauliflower markers, 5.8 between two broccoli cultivars and 7.9 between two cauliflower cultivars. Larger differences for each crop were found between cultivars from different seed companies than within the same company. RAPD markers provide a quick and reliable alternative to identify broccoli and cauliflower cultivars.     

Retrotransposon-based molecular markers for grapevine species and cultivars identification

Insertional polymorphisms of two copia-like (Vine-1, Tvv1) and one gypsy-like (Gret1) retrotransposon found in the grapevine genome were studied in 29 Vitis genotypes (Vitis arizonica, Vitis cinerea, Vitis labrusca, Vitis rupestis, Vitis rotundifolia, Vitis vinifera subsp. sylvestris and 23 V. vinifera subsp. sativa) using inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and sequence-specific amplified polymorphism (SSAP) techniques. IRAP, REMAP and SSAP polymorphisms were compared with amplified fragment length polymorphism (AFLP), Inter-single sequence repeats (ISSR) and SSR polymorphisms by evaluating the information content, the number of loci simultaneously analysed per experiment, the effectiveness of the analyses in assessing the relationship between accessions and the number of loci needed to obtain a coefficient of variation of 10%. The UPGMA dendrograms of each molecular marker system were compared and the Mantel matrix correspondence test was applied. Furthermore, the corresponding insertion ages of the transposable elements were estimated for each retrotransposon subfamily analysed. The presence of Gret1, Tvv1 and Vine-1 retrotransposons in all analysed genotypes suggests that copia-like and gypsy-like retrotransposons are widespread in Vitis genus. The results indicate that these retrotransposons were active before Vitis speciation and contributed to Vitis genus evolution. IRAP, REMAP and SSAP markers allow the discrimination of Vitis species and V. vinifera subsp. sativa cultivars with certainty as has been shown with AFLP, ISSR and SSR analyses, but phylogenetic trees obtained by retrotransposon-based molecular markers polymorphisms show some significant differences in the allocation of the analysed accessions compare to those obtained by ISSR, AFLP and SSR molecular markers. The phylogenetic tree resulting from REMAP polymorphism appeared the most representative of the effective relationship between all analysed accessions.     

Identification of Prunus armeniaca cultivars by RAPD and SCAR markers

Nineteen cultivars of apricot (Prunus armeniaca) were distinguished using random amplified polymorphic DNA (RAPD) markers. One decamer out of 44 used was useful to differentiate cultivars of the Campania Region from those of Northern Italy, North America and Greece. A sequence characterized amplified region (SCAR) marker was obtained. The results provide a protocol to fingerprint DNA of apricots as an efficient way to quality control and fraud prevention.     

Morphological and genetic diversity of the family Azollaceae inferred from vegetative characters and RAPD markers

Family Azollaceae has seven species with a controversial taxonomy. The identification of species without reproductive structures relies on vegetative characters but some are variable, leading to misinterpretations. The molecular methods may be helpful, but until now, they did not provide a conclusive Azolla taxonomy. Therefore, we studied the family Azollaceae at vegetative and molecular levels. Analysis of vegetative, random amplified polymorphic DNA (RAPD) and combined data showed a comparable grouping of the Azolla species in two main clusters: cluster I, referred to as section Rhizosperma (A. pinnata and A. nilotica) and cluster II, referred to as section Azolla (A. filiculoides, A. microphylla, A. caroliniana and A. mexicana), with the exception of A. rubra, which clustered differently depending on the method. All the Azolla species were distinguished by the 13 polymorphic vegetative characters, the 211 RAPD markers or the combined data, with the latest showing the highest discrimination. The Shannon Index diversity was greater with RAPD (2.276) than with vegetative characters (0.054), highlighting the higher discriminating power of the molecular data. The partitioning of diversity was, as expected, high among species for all the types of data and low within species, with the lowest diversity obtained for morphological data. Both data sets (vegetative and RAPD) allowed the distinction of all the species and their clustering into sections Rhizosperma and Azolla, suggesting this as the most correct for this family. The dendrogram from the combined data was the most accurate, highlighting the benefit of integrating different types of data to study the family Azollaceae.     

A first linkage map of olive (Olea europaea L.) cultivars using RAPD,AFLP, RFLP and SSR markers

The first linkage map of the olive (Olea europaea L.) genome has been constructed using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLP) as dominant markers and a few restriction fragment length polymorphisms (RFLP) and simple-sequence repeats (SSR) as codominant markers. Ninety-five individuals of a cross progeny derived from two highly heterozygous olive cultivars, Leccino and Dolce Agogia, were used by applying the pseudo test-cross strategy. From 61 RAPD primers 279 markers were obtained - 158 were scored for Leccino and 121 for Dolce Agogia. Twenty-one AFLP primer combinations gave 304 useful markers - 160 heterozygous in Leccino and 144 heterozygous in Dolce Agogia. In the Leccino map 249 markers (110 RAPD, 127 AFLP, 8 RFLP and 3 SSR) were linked. This resulted in 22 major linkage groups and 17 minor groups with fewer than four markers. In the Dolce Agogia map, 236 markers (93 RAPD, 133 AFLP, 6 RFLP and 4 SSR) were linked; 27 major linkage groups and three minor groups were obtained. Codominant RFLPs and SSRs, as well as few RAPDs in heteroduplex configuration, were used to establish homologies between linkage groups of both parents. The total distance covered was 2,765 cM and 2,445 cM in the Leccino and Dolce Agogia maps, respectively. The mean map distance between adjacent markers was 13.2 cM in Leccino and 11.9 cM in Dolce Agogia, respectively. Both AFLP and RAPD markers were homogeneously distributed in all of the linkage groups reported. The stearoyl-ACP desaturase gene was mapped on linkage group 4 of cv. Leccino.     

Cytogenetic markers for the characterization of Capsicum annuum L. cultivars

Karyotypic studies with conventional staining have been unsuccessful due to the uniformity of Capsicum chromosomes. In this study, we found diagnostic chromosome characters that permit to characterize cultivars; this is the first cytological characterization of both rDNAs (18S and 5S) in a species of Capsicum using a genus-specific probe and the most exhaustive in C. annuum to date. The heterochromatic banding patterns enabled us to identify cultivars, and fluorescent in situ hybridization (FISH) showed one 5S rDNA locus largely conserved within the cultivars, whereas high variation in the number of 18S rDNA loci was observed. One of the most obvious differences is the presence of an additional active nucleolar organizer region in pair #12 and the dispersal of inactive 18S rDNA signals. These results indicate that fluorochrome banding together with silver impregnation and FISH procedures are very useful for the identification of chromosomes and the interpretation of chromosomal variation between cultivars. The functional role of this variation is still uncertain, but our results show that copy number variation of repetitive DNA during the course of evolution might provide an excellent experimental system for studying genome rearrangements accompanying functional divergence in domesticated C. annuum.     

Use of sequence characterised amplified region and RAPD markers in the identification of the white truffle Tuber magnatum Pico

Isolates of white truffles were identified as Tuber magnatum Pico species using a pair of primers selected from a sequence characterised amplified region (SCAR) and a specific random amplified polymorphic DNA (RAPD) marker. The present study reveals that PCR-fragment-pattern polymorphisms, the construction of probes and couples of primers from one or more of these polymorphic fragments may provide a useful and rapid tool for identifying species of ectomycorrhizal fungi in addition to conventional methods (morphological parameters).     

Genetic relatedness of Portuguese almond cultivars assessed by RAPD and ISSR markers

Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyse the genetic diversity of Portuguese Prunus dulcis cultivars and their relationship to important foreign cultivars. Of the primers tested, 6 (out of 60) RAPD and 5 (out of 18) ISSR primers were selected for their reproducibility and high polymorphism. Out of 124 polymerase chain reaction fragments that were scored, 120 (96.8%) were polymorphic. All the plants could be discriminated and constitute a very heterogeneous group. Five unidentified almond plants found in the region of Foz Côa (north Portugal) and wild almond (P. webbii) from Italy and Spain were also included. Four main groups of plants could be distinguished: P. dulcis cultivars; one Foz Côa plant; P. webbii; and P. persica (outgroup). The segregating Foz Côa plant may represent a feral individual or a hybrid between P. dulcis and P. webbii.Abbreviations dNTP Deoxynucleotide triphosphate - CTAB Cetyltrimethylammonium bromide - ISSR Inter-simple sequence repeats - PCR Polymerase chain reaction - RAPD Randomly amplified polymorphic DNA - RASTM Regional Agricultural Services of Trás-os-Montes - TE Tris-EDTA buffer - UPGMA Unweighted pair group method with arithmetical averagesCommunicated by P. Puigdoménech     

Monitoring of cultivar identity in micropropagated olive plants using RAPD and ISR markers

Randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers were applied to assess the genetic stability of micropropagated olive (Olea europaea L. cv. Maurino) plants regenerated by axillary buds. Initial olive explants, isolated from one donor tree, were multiplied on Murashige and Skoog medium for 12 repeated subcultures. A total of 40 RAPD and 10 ISSR markers resulted in 301 distinct and reproducible band classes showing homogeneous RAPD and ISSR patterns. The amplification products revealed genetic stability among the micropropagated plants and between them and the donor plant. The results demonstrate the genetic stability of nine year old mature micropropagated olive plants cultured in field, and corroborated the fact that axillary multiplication is the safest mode for multiplication of true to type plants.     

The use of ISSR and RAPD markers for detecting DNA polymorphism,genotype identification and genetic diversity among barley cultivars with known origin

The potential of bulk analyses of RAPD and ISSR-PCR markers for fingerprinting purposes was evaluated using ten RAPD and ten ISSR primers. The phylogenetic relationships of 16 barley cultivars from different countries, and all having a known pedigree, were analysed using 353 PCR markers (125 RAPDs and 228 ISSRs). The band profiles generated were reproducible in spite of the different DNA extractions, PCR techniques, electrophoretic methods and gel scorings used. The RAPD primer S10 and four ISSR primers (811, 820, 835 and 881) were both able to distinguish all cultivars. A strong and quite linear relationship was observed between Resolving Power (Rp) of a primer and its ability to distinguish genotypes. The dendrograms obtained using these two molecular markers are in agreement with their known origin, showing clusters that separate very well the spring/winter and six-rows/two-rows cultivars. Thus, bulk analyses of RAPD and ISSR PCR markers provides a quick, reliable and highly informative system for DNA fingerprinting and also permit to establish genetic relationships which agree with, by other means, known origin of the cultivars.     

A first linkage map of pecan cultivars based on RAPD and AFLP markers

We report here the first genetic linkage maps of pecan [Carya illinoinensis (Wangenh.) K. Koch], using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. Independent maps were constructed for the cultivars Pawnee and Elliot using the double pseudo-testcross mapping strategy and 120 F₁ seedlings from a full-sib family. A total of 477 markers, including 217 RAPD, 258 AFLP, and two morphological markers were used in linkage analysis. The Pawnee linkage map has 218 markers, comprising 176 testcross and 42 intercross markers placed in 16 major and 13 minor (doublets and triplets) linkage groups. The Pawnee linkage map covered 2,227 cM with an average map distance of 12.7 cM between adjacent markers. The Elliot linkage map has 174 markers comprising 150 testcross and 22 intercross markers placed in 17 major and nine minor linkage groups. The Elliot map covered 1,698 cM with an average map distance of 11.2 cM between adjacent markers. Segregation ratios for dichogamy type and stigma color were not significantly different from 1:1, suggesting that both traits are controlled by single loci with protogyny and green stigmas dominant to protandry and red stigmas. These loci were tightly linked (1.9 cM) and were placed in Elliot linkage group 16. These linkage maps are an important first step towards the detection of genes controlling horticulturally important traits such as nut size, nut maturity date, kernel quality, and disease resistance.     

Genetic variability of introduced and local Spanish peach cultivars determined by SSR markers

A set of 94 peach cultivars including Spanish native peach and foreign commercial cultivars were analyzed using 15 SSR markers, selected for their high level of polymorphism. The number of alleles obtained varied from two to 11 with an average of 6.73 giving 185 different genotypes. All the cultivars showed a unique genetic profile, each one using different genotypic combination of all loci. BPPCT001 was the most informative locus showing also the highest discrimination power. Only six loci allowed the unambiguous separation of all the Spanish native cultivars studied, and the genotypic combination of only eight loci permitted the total differentiation of the 94 peach cultivars analyzed. The six selected loci (BPPCT001, BPPCT006, BPPCT008, PS9f8, UDP98-022, and UDP98-412) seem to be very useful for future Spanish peach identification works, and they will help to establish a molecular data base for native peach cultivars. UPGMA analysis was performed from the genetic distance matrix, and allowed the arrangement of all genotypes according to their genetic diversity. The genetic diversity among cultivars, observed in this work, led to their separation according to their regional origin, their morphological characteristics, and especially according to their fruit traits. Analysis of molecular variance was performed for seven populations from different regions of Spain and USA to examine the distribution of genetic variation of the studied accessions, showing that the major variation occurred within populations in each geographic site. The results reveal the existence of two diversity regions in Spain for peach germplasm.