Chloroplast microsatellite variations in tetraploid alfalfa

Four chloroplast simple sequence repeat (cpSSR) loci were identified in alfalfa (lucerne), Medicago sativa L. The occurrence of allelic variation at these loci was evaluated in 100 plants from 10 populations of tetra‐ploid alfalfa, belonging to four Italian ecotypes and to an Italian (‘Lodi’) and an Egyptian (‘Iside’) variety. Twenty‐four different alleles were identified, four of which were exclusive to the ‘Iside’ variety. The genetic relationship among plants and among populations was analysed by computing an analysis of molecular variance and an unweighted pair group means analysis clustering. This analysis allowed clear separation of the ‘Iside’ variety from Italian germplasm and the recognition of close relationships within the Italian populations. The data presented suggest that cpSSR analysis of tetraploid alfalfa could be used for germplasm polymorphism analysis.     

Extent of RFLP variability in tetraploid populations of alfalfa, Medicago sativa

Seven widely-cultivated alfalfa varieties and three ecotypes adapted to Central Italy were used to evaluate the extent of polymorphism in that species. Twenty plants per accession were analysed with 16 RFLP probes combined with three restriction enzymes (48 probe/enzyme combination in total) and the data were used to compute the Nei's similarity index taken as a measure of inter- and intra-population RFLP variability. The varieties were, in general, more homogeneous than the ecotypes and the cultivars ‘Adriana’ and ‘Florida’ could be differentiated more easily than the others. Few accession-specific hybridizing fragments were scored and seven populations could be distinguished from the others on the basis of significant differences in the frequencies of specific fragments. The DNA of plant populations of several sizes was bulked and the ability to detect a given fragment in pooled samples was related to the fraction of plants having that fragment among the plants forming the bulk. The results are discussed with special emphasis on the practical utilization of RFLPs for varietal identification.     

Estimation of between and within accession variation in selected Spanish melon germplasm using RAPD and SSR markers to assess strategies for large collection evaluation

The population structure of 15 Spanish melon (C. melo L.)accessions, mostly of Group Inodorus, was assessed by the analysis of 16individuals of each accession using 100 random amplified polymorphic DNA (RAPD) bands produced by 36 primers, and allelic variation at 12microsatellite (SSR) loci (23 alleles). A relatively high level of polymorphism (25.6%) was detected using RAPD markers, and eight SSR loci (66.7%) were useful in discriminating accessions. Cluster analysis using RAPD- and SSR-based genetic distance estimates resulted in similar and consistent groupings of most of the accessions studied. The mean genetic distance and standard error among accessions estimated by RAPD variation was 0.421 ± 0.099, and mean SSR-based genetic distance estimate was 0.285 ± 0.141. Albeit many dominant markers examined were fitted to a 3:1 test ratio, deviation from this ratio and from Hardy-Weinberg expectations for many SSR loci suggests that some populations were in genotypic disequilibrium. Moreover, a higher level of genetic variation was observed between Cassaba market classes than within accessions, suggesting that, depending upon the accession, allelic fixation has occurred in these populations. The relatively high level of heterogeneity observed (different band morphotypes and cluster grouping within a particular market class), however, indicates that the Spanish melons examined possess a relatively broad genetic background. An appraisal of accession population structure such as the one reported herein indicates that bulk sampling techniques coupled with molecular analysis techniques that employ a unique array of discriminating markers can provide information leading to effective strategies for diversity analyses of large collections. This revised version was published online in August 2006 with corrections to the Cover Date.     

Organelle based molecular analyses of the genetic relatedness of cultivated alfalfa (Medicago sativa L) to Medicago edgeworthii Sirjaev, and Medicago ruthenica (L.) Ledebour

Medicago edgeworthii Sirjaev and M. ruthenica (L.) Ledebour are allogamous, diploid (2n = 2x = 16) perennials with flat pods.Medicago edgeworthii is indigenous to the Himalayas and alpine areas west to Afghanistan, and Medicago ruthenica is found in Siberia, Mongolia, and Manchuria on open hillsides and mixed grass steppes. Because both species have a remarkable ability to survive extreme cold and poor soils, the possibility of hybridizing them with alfalfa (M. sativa L.) is being investigated. The objective of this research was to conduct an organelle based molecular assessment of the genetic relatedness of cultivated alfalfa (2n = 4x = 32) to M. edgeworthii and M. ruthenica. A hypervariable, intergenic region of cpDNA was amplified, and mtDNA was amplified with two primer pairs developed from soybean (Glycine max L.) mtDNA sequences. Mean Nei and Li genetic distances (GDs) between alfalfa and M. edgeworthii and alfalfa and M. ruthenica were 0.56 and 0.48 (mtDNA), and 0.33 and 0.30 (cpDNA), respectively. Intra specific GDs were 0.37 (mtDNA) and 0.25 (cpDNA) for M. edgeworthii; 0.42 (mtDNA) and 0.15 (cpDNA) for M. ruthenica; and 0 = 0.50 (mtDNA) and 0 = 0.23 (cpDNA) for alfalfa. Cluster analyses grouped someM. edgeworthii and M. ruthenica entries with alfalfa entries. There is some chance that alfalfa and M. edgeworthii entries which clustered closely could be hybridized; chances of alfalfa × M. ruthenica hybridizations appear to be more problematic. This revised version was published online in July 2006 with corrections to the Cover Date.     

Karyotype and C-banding pattern of mitotic chromosomes in alfalfa, Medicago sativa L.

Numerous cytogenetic studies have been performed on alfalfa (Medicago sativa L. 2n= 4x= 32), but only a few have examined the somatic chromosomes. Because the major factor limiting the study of somatic chromosomes of M. sativa is their rather uniform morphology, characterization was attempted using the C-banding technique instead of the traditional Feulgen method. Chromosome morphology and position of the heterochromatic bands resulted in satisfactory characterization of the chromosomes and provided further evidence of the autotetraploid origin of the species. It was possible to identify the homologous chromosomes and arrange them into eight groups of four. The ideogram of the basic genome of eight chromosomes was constructed. The impact of these results on cytogenetic studies and breeding research in alfalfa is discussed.     

Molecular Marker based Genetic Diversity Analysis in Rice (Oryza sativa L.) using RAPD and SSR markers

The availability of an array of molecular marker systems allowed comparing the efficiency of two of these marker systems to estimate the relationships among various taxa. The objective of this study was to assess the genetic diversity among 40 cultivated varieties and five wild relatives of rice, Oryza sativa L. involving simple sequence repeat (SSR) randomly amplified polymorphic DNA (RAPD) markers. The accessions were evaluated for polymorphisms after amplification with 36 decamer primers and 38 SSR primer pairs. A total of 499 RAPD markers were produced among the 40 cultivated varieties and five wild relatives with a polymorphism percentage of 90.0. Out of 38 SSR primer pairs used, only one locus viz., RM115 was monomorphic. The average Polymorphism Information Content (PIC) value was 0.578 and it ranged from a low of zero (RM 115) to a high of 0.890 (RM 202). The Mantel matrix correspondence test was used to compare the similarity matrices and the correlation coefficient was 0. 582. The test indicated that clusters produced based on RAPD and SSR markers were not conserved since matrix correlation value was 0.582 as against the minimum required value of 0.800. The two marker systems contrasted most notably in pair-by-pair comparisons of relationships. SSR analysis resulted in a more definitive separation of clusters of genotypes indicating a higher level of efficiency of SSR markers for the accurate determination of relationships between accessions that are too close to be accurately differentiated by RAPD markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular genetic diversity within and among German ecotypes in comparison to European perennial ryegrass cultivars

Perennial ryegrass (Lolium perenne L.) is the most important grass species for temperate grassland agriculture. The level and distribution of genetic variation in gene bank ecotype collections is still largely unknown but of great interest for the planning of breeding programmes. The objectives of this study were to (i) assess the molecular variance and population structure of German ecotypes at the regional and population level, (ii) assign ecotypes to germplasm pools and (iii) compare the relationship between German ecotypes and previously‐investigated European cultivars of perennial ryegrass, A total of 22 ecotypes originating from three geographic areas in Germany. each with a sample size of 20 individual plants, were investigated with 156 polymorphic RAPD markers. Genetic distance among ecotypes ranged from 0,27 to 0.48, An analysis of molecular variance (AMOVA) revealed a much larger variation within populations (71%) than among them (29%). Ecotypes from North Germany were significantly different from those of South and Middle Germany. Thus, two distinct germplasm pools could be identified. The 22 ecotypes and 22 previously investigated cultivars shared 98% of the molecular variance.     

Evaluating genetic variation and relationships among two bromegrass species and their hybrid using RAPD and AFLP markers

The two most widely grown bromegrass species in North America are smooth bromegrass (Bromus inermis Leyss.), a hay type grass, and meadow bromegrass (Bromus riparius Rehmann), a pasture type grass. Recently a hybrid bromegrass population between the two species has been produced as a dual-purpose hay-pasture grass. Molecular markers have the potential to improve selection procedures to enhance bromegrass breeding. The objective of this study was to use RAPD and AFLP markers to determine genetic relationships and variations among bromegrass populations. Forty-three RAPD markers from 21 primers and 83 AFLP markers from seven primer combinations were used. Both marker types were able to group the individuals into their respective populations. The relationships among the individuals within each of the populations were not similar between the two marker types. Analysis of molecular variance (AMOVA) detected greater within-population variation than among-population variation for both marker types. The highest variation was observed in the hybrid population followed by meadow and then smooth bromegrass. The inter-population distance from both markers indicated that the highest genetic distance was between meadow and smooth bromegrass and lowest between smooth and hybrid bromegrass, which reflect the breeding history of the hybrid population. This study showed that both markers are capable of differentiating bromegrass genotypes into their respective populations, detecting genetic variation and relationships of the populations. Results of this study suggest that these two markers can be used in the future to enhance the current breeding practices in bromegrass, however, AFLP markers would be the marker of choice due to the high number of polymorphic markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pedigree, RAPD and simplified AFLP-based assessment of genetic relationships among Avena sativa L. cultivars

Two molecular marker techniques: RAPD and simplified ^PstIAFLP have been compared in order to decide on, which technique is better suited to genetic characterization of oat (Avena sativa L.) cultivars. It was investigated, if the same pattern of variability is revealed by two approaches and whether the observed molecular variability reflects pedigree-based relationships. Polymorphic RAPD and ^PstIAFLP markers were sufficient to distinguish all analysed cultivars, demonstrating the usefulness of both methods for cultivar identification. Genetic similarity estimates derived from RAPD, simplified ^PstIAFLP and combined RAPD and ^PstIAFLP data were compared with coefficients of parentage (COP). Molecular markers-based mean genetic similarities were considerably greater than mean COP value. Correlation coefficients between COP and genetic similarities calculated from RAPD, ^PstIAFLP and combined molecular data were very low and not significant. A better correlation (0.50) was found between similarity estimates derived from RAPD and ^PstIAFLP markers. Four separate dendrograms were constructed based on pedigree and molecular analyses using a neighbor-joining algorithm (NJ). The dendrograms were compared and found to be topologically different. The results of this study showed, that both molecular techniques can be conveniently used for genetic characterization of oat cultivars, however ^PstIAFLP would be the method of choice due to the higher efficiency and reproducibility. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic control of somatic embryogenesis in alfalfa (Medicago sativa L. cv. Adriana)

Summary In alfalfa (Medicago sativa) regeneration is genotype-specific. In order to study the genetic control of somatic embryogenesis and to constitute a synthetic cultivar characterized by its high regeneration ability, 2 embryogenic plants selected from the cv. Adriana were selfed, intercrossed and also crossed in both directions with 5 non-embryogenic genotypes of the same cultivar.Progenies of all crosses were scored for their regeneration ability and results indicate that somatic embryogenesis is under the control of 2 dominant loci. However some non-embryogenic genotypes prevent regeneration when crossed with embryogenic ones and this characteristic is not under the control of a single dominant gene.When plants chosen for their capacity to regenerate within F₁ and S₁ progenies were freely intercrossed the regeneration efficiency dropped to 2% (1 plant out of 50). This result indicates that if the genetic background of the population is changed the regeneration is greatly affected and therefore some other mechanism could play a role in determining plant regeneration.     

RAPD variation within and among populations of globe artichoke cultivar 'Spinoso sardo'

Globe artichoke is a vegetable native to the Mediterranean basin. Its commercial production is mainly based on perennial cultivation of vegetatively propagated clones, which are highly heterozygous and segregate widely when progeny‐tested. The aim of this study was to measure genetic variation using random amplified polymorphic DNA (RAPD) in five populations of ‘Spinoso sardo’, the most widely grown and economically important cultivar in Sardinia (Italy). Analysis of molecular variance (amova ) gave highly significant differences between populations (28.1% of the total genetic diversity); substantial within‐population variation was detected (71.8% of total genetic diversity). The RAPD results show the need to apply clonal selection to narrow the broad genetic variability within the cultivar and to address important questions on the most suitable strategies for germplasm preservation.     

Assessment of genetic relationships among Cucumis spp. by SSR and RAPD marker analysis

The first successful production of a sterile interspecific hybrid obtained from a cross between Cucumis hystrix Chakr. (2n = 2x = 24) and Cucumis sativus var. sativus L. (2n =2x = 14), and its subsequent fertility restoration through chromosome doubling provide an effective means for investigating genetic relationships among Cucumis spp. In this study, random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to investigate relationships among C. s. var. sativus L., C. s. var. hardwickii (R.) Alef., C hystrix, C. hytivus Chen & Kirkbride (the amphidiploid species from chromosome doubling of the C. sativus x C. hystrix interspecific hybrid, 2n = 38), C. melo (2n =2x = 24) and C. metuliferus Meyer and Naudin (2n =2x= 24). A total of 109 SSR bands and 398 RAPD primed sites were used to calculate Jaccard's distance coefficients for cluster analysis using a unweighted pair‐group method using an arithmetic averaging (UPGMA) algorithm. The genetic relationships identified using SSR and RAPD markers were highly concordant, such that the correlation between SSR and RAPD genetic distance (GD) estimates was r = 0.94. SSR and RAPD analysis of 22 accessions allowed for their grouping into two distinct groups designated as CS and CM. While group CS consisted of 11 C. sativus genotypes, and the C. hytivus and C. hystrix accessions, group CM included six C. melo genotypes and C. metuliferus. The GD values between C. hystrix and C. sativus ascribed by SSR and RAPD matrices were 0.59 and 0.57, respectively. These GDs were smaller than those detected between C. hystrix and C melo (0.87 and 0.70 derived from SSR and RAPD markers, respectively).     

Italian rice varieties: historical data, molecular markers and pedigrees to reveal their genetic relationships

Two molecular marker approaches [amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR)] were employed to study genomic relationship among 96 rice cultivars. These included most of the best reputed Italian accessions. AFLP produced 461 fragments, 248 (53%) of which were polymorphic, SSR produced four to 11 alleles in the 12 genomic loci investigated. Genomic similarity was estimated independently for the two molecular marker techniques. Both AFLP and SSR dendrograms agree in splitting the cultivars into two main clusters: a small one, comprising four exotic accessions, and a larger one which could be split into four subgroups. These were also analysed on the basis of historical and pedigree information. This is the first report on the application of DNA polymorphism analysis to reveal genomic relationship among cultivated Italian rice germplasm. Results will be useful for breeding programmes.     

Assessment of natural and induced genetic variation in Alstroemeria using random amplified polymorphic DNA (RAPD) markers

Summary We have used random amplified polymorphic DNA (RAPD) markers to study genetic variation in Alstroemeria. The first objective was to examine the discriminatory power of RAPD markers in different genotypes of Alstroemeria obtained by traditional breeding. All genotypes examined, including commercial Alstroemeria varieties, could be distinguished on the basis of their RAPD profiles. Progeny plants could be distinguished from their parents. A second objective of this study was to investigate whether RAPD markers can be used as a routine tool to detect mutant plants, as an alternative to glasshouse testing. To address this objective, we analysed Alstroemeria plants that carried phenotypically visible mutations that either were induced by irradiation using X-rays or were the result of somaclonal variation. In eight out of a total of 13 mutant Alstroemeria plants obtained after irradiation or tissue culture we detected no polymorphisms when compared to control plants that were considered to be non-mutated. Only in five of the mutant plants analysed we detected one to two polymorphisms. These results suggest that frequent genome rearrangements had not occurred in the mutant plants analysed. These results also demonstrate that the RAPD technique is an inappropriate tool for the rapid screening of Alstroemeria for induced variation. It that the RAPD technique is an inappropriate tool for the rapid screening of Alstroemeria for induced variation. It seems probable that this conclusion would be equally applicable in other plant genera in which induced variation has occurred. However, the RAPD technique is a simple and effective tool for genetic fingerprinting of Alstroemeria varieties, provided their differences are due to sexual propagation.     

Comparing RAPD and AFLPTM analysis in discrimination and estimation of genetic similarities among apple (Malus domestica Borkh.) cultivars

Forty-one apple (Malus × domestica Borkh.) cultivars were screened for RAPD (Random Amplified Polymorphic DNA) and AFLP(Amplified Fragment Length Polymorphism) markers. RAPD analysis was performed with 35 arbitrary 10-mer primers, selected from 60 primers tested (kits A, C and E, Operon Technologies, Inc.). Of a total of 362bands observed, 208 (57.5%) were polymorphic. Three-hundred-and-eighty-one AFLP fragments were obtained with 8primer combinations, of which 218 (57.2%) were polymorphic. Cultivars differentiated through mutation were included in this study and showed identical patterns when analysed with both RAPD and AFLP analysis. The estimated genetic relationships were correlated (r = 73.7%) between the analysis with the two different markers. UPGMA analysis was performed and dendrograms were constructed using either the data apart from each(RAPD and AFLP) method or combined in a single joint matrix. The relationships among the forty-one studied cultivars were basically consistent with the known lineage and geographic origins of the cultivars. The four Portuguese cultivars included in this study clustered together and diverged from the other cultivars. Apparently they constitute an independent genetic pool, which could be of interest for apple plant breeders. This revised version was published online in July 2006 with corrections to the Cover Date.     

AFLP, ISSR and RAPD markers reveal high levels of genetic diversity among Lupinus spp.

The Lupinus genus includes a number of important crop species. The use of defined nucleotide sequences for the analysis of genetic diversity among these species has revealed modest levels of diversity. The aim of this study was to access AFLP, ISSR and RAPD markers to evaluate the genetic diversity among L. albus, L. angustifolius, L. cosentinii, L. hispanicus, L. luteus, L. mutabilis, L. pilosus and L. polyphyllus. Unexpectedly, low levels of genetic similarity were found (ranging from 0.205 to 0.432), regardless of the type of molecular marker used. Nevertheless, these techniques consistently showed a greater genetic similarity between L. pilosus and L. cosentinii, L. mutabilis and L. polyphyllus and among L. luteus, L. hispanicus and L. angustifolius, clearly separating the Old World from the New World species. Such low genetic similarity among Lupinus spp. is most unlikely to be due to differences in coding sequences but could be the result of a long diverging process concerning non‐coding regions, which would represent a very important proportion of these genomes.     

Evaluation of genetic variation in Lachenalia bulbifera (Hyacinthaceae) using RAPDs

RAPD (randomly amplified polymorphic DNA) analyses were carried out on 21 accessions of Lachenalia bulbifera (Cyrillo) Engl. Five pre-selected primers produced an average of 88% polymorphisms. Fifteen of the 21 accessions could be identified using the five primers. In a pairwise comparison genetic distance values ranging from 0.11 to 1.08 were obtained. These values reveal a high amount of variation within the species. The genetic distance values within the tetraploid and hexaploid groups on the south coast were low, but values were high between the groups on the south coast and those on the west coast. A dendogram was constructed from the RAPD banding profiles, using UPGM cluster analysis. The dendogram clusters certain accessions together. These clusters are supported by their geographical locality and chromosome data. The hexaploid group, tetraploid group and octoploid group on the south coast are respectively clustered together. It is concluded that RAPDs can be used to assess the genetic variation at an intra-specific level in Lachenalia. This revised version was published online in July 2006 with corrections to the Cover Date.     

锌锰微肥对苜蓿生产的效应研究

针对河南省气候土壤特点、锌锰钼肥的生理功能以及牧草生产利用中存在的问题,对Zn、Mn两种微肥在紫花苜蓿（Medicago sativa L.）优质牧草生产中的配施技术进行了研究。结果表明：紫花苜蓿的有效范围为400~500mg/kg,最佳浓度为430mg/kg。430mg/kg的Zn肥与380mg/kg的Mn 肥结合使用,效果更好,可使牧草产量提高约10.7%,种子产量提高13.7%。