RFLP analysis allows for the identification of alfalfa ecotypes

Three seed lots, obtained from different ‘foundation farms’, for each of the alfalfa, Medicago sativa L., ecotypes ‘Vogherese’ and ‘Maremmana’, together with the variety ‘Iside’ as a control, were studied to test the possibility of distinguishing them through restriction fragment length polymorphism analysis. Twenty plants per seed lot were analysed with 25 DNA probes that yielded 155 informative polymorphic fragments. Variance partitioning showed that within‐population variability accounted for nearly 98% of the total variance, while a small but significant contribution to the total variance was due to among‐lot variability.‘Vogherese’ was more homogeneous than ‘Maremmana’, as a consequence of the greater environmental homogeneity of the adaptation area of the former ecotype compared with the latter. Bulk analysis yielded eight variety‐specific bands, 12 bands present in both ecotypes and absent in the variety and one band specific of ‘Maremmana’. Results are discussed in relation to the practical use of molecular markers for alfalfa ecotype identification.     

Alfalfa germplasm from a Sahara oasis: characterization by means of bio-agronomic traits and SSR markers

In North African oases Medicago sativa ssp. sativa is likely to have been cultivated for long time without contacts with ssp. falcata . In order (i) to verify the genetic distinctiveness of the alfalfa germplasm of the oasis and (ii) to examine their internal variation, 17 farm landraces were collected in Siwa oasis (Egypt) and studied with four Egyptian and three Italian cultivars (120 plants/population) by means of bio-agronomic characters in 2 years cultivation and SSR markers in 'bulk' samples. Siwa germplasm was similar to the cultivars for dry matter yield but differed for plant form, earliness and reaction to fall conditions; SSR analysis based on 31 polymorphic DNA fragments distinguished Siwa landraces from the two groups of varieties. The variation within Siwa group was of continuous type based on bio-agronomic traits; average genetic similarity was high (0.8959) and UPGMA dendrogram suggests poor isolation among the landraces, although all, except two, were uniquely fingerprinted. The use of putative 'pure' subspecies of high agronomic value in the alfalfa variety construction process is discussed.     

Genetic diversity and phylogenetic relationships among accessions of hop, Humulus lupulus, as determined by amplified fragment length polymorphism fingerprinting compared with pedigree data

DNA fingerprinting using amplified fragment length polymorphisms (AFLPs) was successfully employed to detect genetic relationships and variability among 90 hop cultivars and breeding lines comprising a collection of the world's hop germplasm. Seven AFLP primer combinations produced a total of 347 fragments of which 151 (43.5%)) were polymorphic. One‐hundred and thirty informative, highly reproducible DNA polymorphisms were used to estimate the genetic similarity (GS) which varied between 1.0 (e.g. ‘Saazer’ vs. ‘Tettnanger’) and 1.17 (‘Columbus’ vs. ‘Tettnanger’, ‘Spalter’ and ‘Saazer’). UPGMA (unweighted pair‐group method with arithmetic averages) clustering revealed two main clusters, reflecting the two main sources of origin and the two main breeding objectives: one cluster of mainly European origin representing the aroma pool and a second cluster associating accessions with European germplasm infiltrated by wild American genes with less aroma quality, but a higher bittering potential. Each main branch was composed of four or three subclusters with subgroups, respectively. Assignment of almost all genotypes in the dendrogram was consistent with the pedigree data as far as they are known. Consequently, AFLPs are shown to be suitable for assessing the genetic variability in hop germplasm and are useful for describing the genetic relationships among cultivars and accessions, which allows phylogenetic questions to be addressed.     

Genetic diversity assessment in cultivated cardoon by AFLP (amplified fragment length polymorphism) and microsatellite markers

Cultivated cardoon (Cynara cardunculus L. var. altilis DC) belongs, together with globe artichoke (C. (cardunculus L. var. sylvestris L.) and wild cardoon (C. cardunculus L. var., sylvestris (Lamk) Fiori). to the family Asteraceae (Compositae). Cultivated cardoon is of regional importance in Italy. Spain and southern France, where it is used for the preparation of traditional dishes. It has been shown to have potential as a source of oil from its seeds, inulin from its roots and various biopharmaceuticals from its leaves. Levels of genetic diversity and relatedness between eleven Italian and 10 Spanish accessions were assessed by DNA profiling with eight AFLP primer combinations and at five microsatellite loci. The AFLP analysis of genetic similarities showed that the Spanish and Italian accessions represent two distinct gene pools; substantial variation was present within each accession. On the other hand a limited variation was detectable by applying SSR markers.     

Identification of commercial chicory cultivars for hydroponic forcing and their phenetic relationships revealed by random amplified polymorphic DNAs and amplified fragment length polymorphisms

One‐hundred and twenty‐four amplified fragment length polymorphism (AFLP) and 49 random amplified polymorphic DNA (RAPD) markers have been used to distinguish between 20 and 23 commercial chicory cultivars, respectively. These were all Cichorium intybus var. foliosum F₁ hybrids, currently used in hydroponic forcing. Five‐hundred and twenty RAPD primers (OPERON) were tested, of which 156 resulted in reproducible patterns and 26 yielded polymorphisms. Two‐hundred and fifty‐six AFLP primer‐combinations were tested and six combinations were selected for identification purposes. Similarity indices were measured and clustering has been done using pairwise comparison. Both types of marker provide similar conclusions. Two major clusters are formed, representing late and early cultivars. All cultivars were identified using 10 informative RAPD primers or three AFLP primer combinations. A low degree of polymorphism was detected between some early cultivars, suggesting a narrow genetic base in their breeding strategy.     

Evaluation of amplified fragment length polymorphism markers in tef, Eragrostis tef (Zucc.) Trotter, and related species

Tef is an important cereal crop in Ethiopia. This study was conducted to investigate (1) genetic diversity within and among three Eragrostis species (E. tef, E. pilosa and E. curvula), and (2) the relationship between E. tef, E. pilosa and E. curvula. A total of 529 AFLP markers were obtained, out of which 58% (368) were polymorphic, using 10 primer, combinations. The three species were separated distinctly using amplified fragment length polymorphism (AFLP), However, diversity revealed at the morphological trait level was not commensurate with that observed for AFLP. This was due to the small number of morphological traits available and their interaction with the environment. Within tef, ‘Rubicunda’ and DZ‐01‐1093 were found to be distantly related to the rest of the tef accessions. The diversity within species was such that E. pilosa was the most diverse followed by E. curvula and E. tef. Moreover, E. pilosa was more closely related to E. tef than E. curvula. Therefore, further study is needed of E. pilosa accessions and of ‘Rubicunda’ and DZ‐01‐1093 in a crossing programme to generate a population for selection and/or genetic mapping. A total of 19 cultivars or accessions had one or more unique fragments using one or more AFLP primers, indicating the potential of the technology in fingerprinting tef in a breeding or seed multiplication programme.     

Verification of the parthenogenetic capability of unreduced eggs in an alfalfa mutant by a progeny test based on morphological and molecular markers

Apomixis involves the parthenogenetic development of apomeiotic eggs. It has the potential of cloning plants through seed, and thus furnishes a unique opportunity in breeding of allogamous sexual species, such as alfalfa, for developing superior cultivars with permanently fixed heterosis. Apomixis as a whole has not been detected in the genus Medicago, but components of apomixis have been reported. The formation of unreduced eggs through diplosporic meiosis was documented in a diploid mutant of M. saliva ssp. falcata (L.) Arcang., named PG-F9. Since in facultative apomictic species non-reductional meiosis and parthenogenesis could be tightly associated processes, a progeny test based on morphological trait and molecular marker evaluation was carried out to verify the occurrence of parthenogenesis in PG-F9. Morphological traits such as leaf shape, stipule form, stem pigmentation and flower colour were shown to be effective in the preliminary screening of progenies and most of the plants were classified as non-maternal (i.e. from sexual reproduction). Molecular investigations by means of random amplified polymorphic DNA (RAPD) fingerprint and heterozygous restriction fragment length polymorphism (RFLP) loci detection conducted on the progenies classified morphologically as maternal allowed two plants, molecularly similar but not identical to PG-F9, to be discovered. Owing to the high number of molecular markers conserved as in the mother plant, and because of the great discriminating efficiency of the primers and probes used, these progeny plants could most likely be generated through parthenogenesis of diplosporic eggs. In fact, the extraordinary preservation of maternal morphological traits and genomic loci over one generation may be explained only if apomictic reproductive events rarely took place in PG-F9.     

Estimating genetic relationships among historical sources of alfalfa germplasm and selected cultivars with sequence related amplified polymorphisms

Fifteen alfalfa populations consisting of six public cultivars and nine historically recognized sources of alfalfa germplasm in North American cultivars were examined using sequence related amplified polymorphisms (SRAPs). Three bulk DNA samples from each population were evaluated with fourteen different SRAP primer pairs. This resulted in 249 different amplicons, of which over 90% were polymorphic. A dendrogram from the analysis suggests that the public cultivars are quite diverse from all the historical sources of germplasm. The highest mean genetic similarity among the nine original sources of Medicago germplasm was 0.85 between PI 536535 (Peruvian) and 536536 (Indian), while the lowest (0.47) was between PI 560333 (M. falcata) and 536539 (African). The highest mean genetic similarity among the nine original sources of Medicago germplasm and the public alfalfa cultivars was 0.78 between PI 536532 (Ladak) and Vernal, while the lowest (0.59) was between PI 536539 (African) and Oneida. Relationships based on SRAP analysis appear to generally concur with expected relationships based on fall dormancy. This report demonstrates that SRAPs are a promising marker system for detecting polymorphisms in alfalfa.     

Use of RAPD and microsatellite (SSR) variation to assess genetic relationships among populations of tetraploid alfalfa, Medicago sativa

The level of random amplified polymorphic DNA (RAPD) and microsatellite variation present in four ecotypes and two varieties of alfalfa (lucerne) from Italian and Egyptian germplasm sources was evaluated. A sample of 100 plants from 10 populations was analysed by means of 41 RAPD markers and 37 simple sequence repeat (SSR) markers. Both molecular approaches revealed a high degree of genetic diversity within each of the cultivated populations and enabled each of the plants considered to be uniquely fingerprinted. The genetic relationships among plants and populations were analysed by computing AMOVA (analysis of molecular variance) and F_ST analyses. RAPDs were able to separate the Italian populations from the Egyptian variety. SSRs allowed strong separation of the four Italian alfalfa ecotypes. It was concluded that RAPD and microsatellites could be useful and powerful tools for assessing genetic variation and genetic relationships in tetraploid alfalfa.     

Amplified fragment length polymorphism as a tool for molecular characterization of almond germplasm: genetic diversity among cultivated genotypes and related wild species of almond,and its relationships with agronomic traits

Amplified fragment length polymorphism (AFLP) analysis is a rapid and efficient method for producing DNA fingerprints and molecular characterization. Our objectives were to: estimate genetic similarities (GS), marker indices, and polymorphic information contents (PICs) for AFLP markers in almond cultivars; assess the genetic diversity of almond cultivars and wild species, using GS estimated from AFLP fingerprints and molecular characterization; and facilitate the use of markers in inter-specific introgression and cultivar improvement. The genetic diversity of 45 almond cultivars from Iran, Europe, and America, were studied assaying 19 primer combinations. In addition, several agronomic traits were evaluated, including flowering and maturity times, self-incompatibility, and kernel and fruit properties. Out of the 813 polymerase chain reaction fragments that were scored, 781 (96.23%) were polymorphic. GS ranged from 0.5 to 0.96, marker indices ranged from 51.37 to 78.79, and PICs ranged from 0.56 to 0.86. Results allowed the unique molecular identification of all assayed genotypes. However, the correlation between genetic similarity clustering as based on AFLP and clustering for agronomic traits was low. Cluster analysis based on AFLP data clearly differentiated the genotypes and wild species according to their origin and pedigree, whereas, cluster analysis based on agronomic data differentiated according the pomological characterization. Our results showed the great genetic diversity of the almond cultivars and their interest for almond breeding.     

Associations among widely used French and U.S. maize hybrids as revealed by restriction fragment length polymorphisms

Summary Eighty DNA Restriction Fragment Length Polymorphism (RFLP) clones were used as probes to profile 47 hybrids of maize (Zea mays L.) that are of widespread usage in France and 49 hybrids that are either in common usage or are new releases in the U.S. The objectives were to 1) investigate the degree to which RFLPs provide unique characterization of hybrids; 2) show associations among hybrids using both cluster and principal coordinate analyses; 3) measure the ability of RFLPs to show associations among hybrids that reflect those to be expected on the basis of pedigree; and 4) compare the patterns and extent of genetic diversity among French hybrids with that found among a set of widely used U.S. hybrids.RFLPs showed all French hybrids to have different profiles, however, 3 hybrids were very similar with more than 90% of their profiles in common. Twenty-seven U.S. hybrids showed this level of similarity with one or more U.S. hybrids. High correlations (r=0.93, 0.94) were found for pedigree distance versus RFLP distance between pairs of French and of French and U.S. hybrids, respectively. Similar levels of correspondence for rank correlations between RFLP and pedigree data were also found. Similar groupings of hybrids were shown by two cluster analysis methods and by principal coordinate analysis. Inclusion of hybrids in cluster groupings was supported by observation of raw distance data for selected hybrids and their nearest neighbors. Most hectarage in France is planted to hybrids that fall within 2 related groups of germplasm on the basis of RFLP data. Minimum distance standards could promote breeders to surmount the challenge of introducing elite yet diverse germplasm into agriculture.     

Genetic diversity and identification of cymbidium cultivars as measured by random amplified polymorphic DNA (RAPD) markers

DNA from thirty-six cymbidium cultivars was examined using polymerase chain reaction (PCR) to determine the efficiency of randomly amplified polymorphic DNA (RAPD) markers in identifying cultivars and determining levels of genetic variability. A total of 132 RAPD markers, 78% of which were polymorphic, were produced from 15 10mer arbitrary primers. All the cultivars were distinguishable when a number of primers was considered. One cultivar, Blue Smoke ‘Green Meadow’ could be distinguished from all the rest based only on lack of the OPA5-370 fragment. Genetic distances among the cultivars were estimated based on the amount of band sharing and ranged from 0.08–0.50 with an average of 0.29. Cluster analysis of genetic distance estimates grouped siblings together with each other and parents with offsprings, thereby agreeing with known parentage information and corroborating isozyme data obtained from a separate study. The possible application of the observed polymorphism and variation to cymbidium breeding is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic variation and cultivar identification of Jamaican yam germplasm by random amplified polymorphic DNA analysis

Summary We have used random amplified polymorphic DNA (RAPD) analysis to characterize eleven cultivars of the five economically most important yam species grown in Jamaica (Dioscorea alata, D. cayenensis, D. rotundata, D. trifida and D. esculenta). Amplification of genomic DNA samples with nine different arbitrary 10mer primers revealed a total of 338 different band positions, ranging in size from 0.3 to 2.5 kb. RAPD patterns proved to be highly reproducible and somatically stable. While no variation was observed among plants belonging to the same cultivar, a large number of intervarietal and interspecific polymorphisms enabled us to reliably discriminate between all Jamaican cultivars investigated.     

Genetic characterization and identification of new accessions from Syria in an olive germplasm bank by means of RAPD markers

Thirty-two olive cultivar accessions from Syria, most of them obtained from collecting expeditions, were characterized by means of RAPD markers before being introduced in the World Germplasm Bank of Cordoba. A total of 79 polymorphic bands(6.1 polymorphisms per primer) out of 93(7.1 bands per primer) were scored for the13 primers used, corresponding to 84.9% of the amplification products. Thirty-one different genotypes were clearly discriminated. Differences were not found among the amplification profiles from different individuals of the same cultivar. Only two cases of mislabeling or errors of planting were found. Fourteen accessions corresponding to 6 homonyms were discriminated by RAPDs as different genotypes. The dendrogram obtained by RAPD analysis included three major groups. Some evidence of relationships of the Syrian accessions studied according to their geographic origin and/or diffusion was found. For instance, cultivars from the Central Syria (Tadmur/Palmyra)such as Toffahi', ‘Abbadi Abo Gabra’-1033,‘Abo Kanani’, ‘Shami’-1041, ‘Abbadi Shalal’ ‘Adgam’-844 and ‘Majhol’-1013 clustered in Group 1 and 2. Six cultivars from Northern Syria clustered in Group 2. But it was not found a geographic structure for the cultivars from South and West of Syria. These results agree with the hypothesis of autochthonous origin of most of the olive cultivars. Some associations between cultivars from Central Syria and their fruit size were observed. This suggests that fruit size was a criterion of local selection in olive cultivars of this area. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of resistance genes against scald (Rhynchosporium secalis (Oudem.) J.J. Davis) in barley (Hordeum vulgare L.) lines from central Norway, by means of genetic markers and pathotype tests

Rhynchosporium secalis is a serious pathogen of barley (Hordeum vulgare L.) in central Norway. A breeding effort was initiated in 1977 to introduce resistance from different sources into adapted genotypes, and the first cultivar from the program was recently released. However, little is known about the resistance genes introgressed in this cultivar or in advanced breeding lines. An effort was made to address this issue through a set of isolates and available molecular markers. Fourteen breeding lines and their resistance donors were investigated by evaluating their reactions to 11 R. secalis isolates. Bulked segregant analysis was used to identify molecular markers linked to resistance genes in 12 of the breeding lines. The isolates were found to be of less discriminating value than the markers. Useful information has been obtained as to the nature of several of the resistance genes introgressed. Eight of the 12 breeding lines contained introgressed genes that were located at the `complex Rh' locus on chromosome 3H and hence may not easily be pyramided into the same genotype. Previous information about the nature of the resistance in `Jet' is questioned. Neither of the resistance genes Rh or Rh2 seems to have been incorporated into Norwegian breeding material.     

Genetic diversity of Brassica napus L. Germplasm from China and Europe assessed by some agronomically important characters

Summary The genetic diversity and relationships among 63 rapeseed accessions, including 34 Chinese, 22 Czech, 2 Swedish, 2 German, one French and 2 Canadian accessions, were evaluated by nine agronomically important characters in the field at Yangling, Shaanxi, China. Significant differences between Chinese and European group in plant height, setting position of the first primary branch, number of siliques of the terminal raceme, thousand seed weight and seed yield per plant were detected. There were significant variations in nine agronomic characters among the tested rapeseed accessions. Ward’s minimum variance cluster analysis based on Mahalanobis distances on the raw data of nine agronomic characters clearly separated the European accessions from the Chinese ones. However, the Chinese accessions with erucic acid free and/or low glucosinolates could not be separated from those Chinese accessions with both high erucic acid and glucosinolates. In general, cluster analysis of the 63 accessions based on the selected agronomic characters was consistent with known pedigree information and geographic origin, as well as the previous RAPD results of these accessions. The European rapeseed could be important germplasm resources for enriching the genetic background of Chinese rapeseed, and vice versa.