Molecular characterization of the genetic integrity of wheat (Triticum aestivum L.) germplasm after long-term maintenance

The genetic identity of eight wheat (Triticum aestivum L.) accessions maintained in the Gatersleben genebank and regenerated up to 24 times was studied by using wheat microsatellite markers (WMS). It was demonstrated that WMS can be used to analyze bulks of seeds stored more than 50 years in a seed reference collection at room temperature. No contamination due to foreign pollen or incorrect handling during the multiplication cycles was discovered. For one accession (TRI 4599) genetic drift was observed, whereas for TRI 249 a heterogenous situation for two markers was maintained over the years. We were able to show that microsatellites can be used as a simple and reliable marker system for the verification of the integrity and genetic stability of genebank accessions. Received: 29 March 1999 / Accepted: 22 June 1999     

Assessment of genetic diversity within and among germplasm accessions in cultivated sorghum using microsatellite markers

Microsatellite markers are increasingly being used in crop plants to discriminate among genotypes and as tools in marker-assisted selection. Here we evaluated the use of microsatellite markers to quantify the genetic diversity within as well as among accessions sampled from the world germplasm collection of sorghum. Considerable variation was found at the five microsatellite loci analysed, with an average number of alleles per locus equal to 2.4 within accessions and 19.2 in the overall sample of 25 accessions. The collection of sorghum appeared highly structured genetically with about 70% of the total genetic diversity occurring among accessions. However, differentiation among morphologically defined races of sorghum, or among geographic origins, accounted for less than 15% of the total genetic diversity. Our results are in global agreement with those obtained previously with allozyme markers. We were also able to show that microsatellite data are useful in identifying individual accessions with a high relative contribution to the overall allelic diversity of the collection. Received: 10 August 1999 / Accepted: 27 August 1999     

Microsatellite analysis of Aegilops tauschii germplasm

The highly polymorphic diploid grass Aegilops tauschii isthe D-genome donor to hexaploid wheat and represents a potential source for bread wheat improvement. In the present study microsatellite markers were used for germplasm analysis and estimation of the genetic relationship between 113 accessions of Ae. tauschii from the gene bank collection at IPK, Gatersleben. Eighteen microsatellite markers, developed from Triticum aestivum and Ae. tauschii sequences, were selected for the analysis. All microsatellite markers showed a high level of polymorphism. The number of alleles per microsatellite marker varied from 11 to 25 and a total of 338 alleles were detected. The number of alleles per locus in cultivated bread wheat germplasm had previously been found to be significantly lower. The highest levels of genetic diversity for microsatellite markers were found in accessions from the Caucasian countries (Georgia, Armenia and the Daghestan region of Russia) and the lowest in accessions from the Central Asian countries (Uzbekistan and Turkmenistan). Genetic dissimilarity values between accessions were used to produce a dendrogram of the relationships among the accessions. The result showed that all of the accessions could be distinguished and clustered into two large groups in accordance with their subspecies taxonomic classification. The pattern of clustering of the Ae. tauschii accessions is according to their geographic distribution. The data suggest that a relatively small number of microsatellites can be used to estimate genetic diversity in the germplasm of Ae. tauschii and confirm the good suitability of microsatellite markers for the analysis of germplasm collections. Received: 8 September 1999 / Accepted: 7 October 1999     

Onion microsatellites for germplasm analysis and their use in assessing intra- and interspecific relatedness within the subgenus Rhizirideum

We have identified a set of informative STMS markers in onion (Allium cepa L.) and report on their application for genotyping and for determining genetic relationships. The markers have been developed from a genomic library enriched for microsatellites. Integrity of the microsatellite polymorphism was confirmed by amplicon sequencing. The microsatellite genotypes of 83 onion accessions and landraces from living onion collections were compared. As few as four primer pairs were sufficient to assign unique microsatellite patterns to the 83 accessions. Some of the microsatellite markers can be used for interspecific taxonomic analyses among close relatives of Allium cepa. Generally, our data support and extend results obtained from recently performed analyses using ITS, RAPD and morphology. Received: 8 October 1999 / Accepted: 3 November 1999     

Assessment of genetic diversity in a Morus germplasm collection using fluorescence-based AFLP markers

To meet various breeding objectives and to conserve the existing genetic resources of mulberry for future use, the present study was undertaken to investigate the amount of genetic diversity and to establish the relationships between mulberry genotypes using fluorescence-based AFLP markers. Genetic diversity was estimated in 45 mulberry accessions from different eco-geographic regions of Japan and other parts of the world. Five primer combinations amplified an average of 110 AFLP markers per primer combination, ranging in size from 35 to 500 bp. A high degree of polymorphism was revealed by these combinations that ranged from 69.7 to 82.3% across all the genotypes studied. Several rare genotype-specific bands were also identified which could be effectively utilized to distinguish different genotypes. The wide range in genetic similarity coefficients (0.58–0.99) indicated that the mulberry germplasm collection represents a genetically diverse popu-lation. The phenetic dendrogram generated by the UPGMA method grouped 45 accessions into four major clusters, which was in agreement with the results from conventional methods. Clustering of some genotypes into strictly separate groups was not readily apparent and no clear interrelationships could be depicted, in spite of their different geographic origin. In addition, AFLP analysis provided sufficient polymorphism for DNA typing and contributed additional insights into the genetic structure of the mulberry germplasm. These results will help in the formulation of appropriate strategies for conservation and variety improvement in mulberry, for which little or no knowledge of genetic diversity is currently available. Received: 30 December 1999 / Accepted: 14 March 2000     

Simple sequence repeats for the genetic analysis of apple

 The development of highly informative markers, such as simple sequence repeats, for tagging genes controlling agronomic characters is essential for apple breeding. Furthermore the use of these markers is fundamental both for variety identification and for the characterisation and management of genetic resources. We have developed 16 reliable simple sequence repeat (SSR) markers that amplify all alleles from a panel of 19 Malus x domestica (Borkh.) cultivars or breeding selections and from Malus floribunda 821. Those markers show a high level of genetic polymorphism, with on average 8.2 alleles per locus and an average heterozygosity of 0.78. Due to this high level of polymorphism, it was possible using two selected SSRs to distinguish all cultivars except Starking and Red Delicious. Ten of the markers we developed have been mapped on a RAPD linkage map, proving their Mendelian segregation as well as their random distribution in the apple genome. Finally, we discuss the importance of using co-dominant markers in outbreeding species. Received: 8 October 1997 / Accepted: 9 December 1997     

The use of microsatellites for germplasm management in a Portuguese grapevine collection

To initiate the characterization of the Portuguese grapevine genepool, we have genotyped 49 Portuguese grapevine cultivars at 11 microsatellite loci. The markers proved to be informative in the Portuguese cultivars, with expected heterozygosity ranging from 0.67 to 0.84. At most loci, an excess of heterozygous individuals was observed, while the deficiency of heterozygotes at 1 locus (VVMD6) indicated the presence of null alleles. On the basis of the microsatellite allele data several previously assumed synonyms were verified: (1) ’Fernão Pires’=’Maria Gomes’, (2) ’Moscatel de Setúbal’=’Muscat of Alexandria’, (3) ’Boal Cachudo’=’Boal da Madeira’=’Malvasia Fina’, (4) ’Síria’=’Crato Branco’= ’Roupeiro’ and (5) ’Periquita’=’Castelão Francês’=’João de Santarém’=’Trincadeira’. Although the three varieties ’Verdelho da Madeira’, ’Verdelho dos Açores’, and ’Verdelho roxo’ are regarded by the Lista Nacional de Sinónimos as distinct cultivars, they displayed identical SSR profiles at 17 loci and appear to represent types of 1 single cultivar. The genetic profiles of all 49 cultivars were searched for possible parent-offspring groups. The data obtained revealed the descendence of ’Boal Ratinho’ from ’Malvasia Fina’ and ’Síria’.     

Microsatellite variability in grapevine cultivars from different European regions and evaluation of assignment testing to assess the geographic origin of cultivars

Nine microsatellite markers (VVMD5, VVMD7, VVS2, ssrVrZAG21, ssrVrZAG47, ssrVrZAG62, ssrVrZAG64, ssrVrZAG79 and ssrVrZAG83) were chosen for the analysis of marker information content, the genetic structure of grapevine cultivar gene pools, and differentiation among grapevines sampled from seven European vine-growing regions (Greece, Croatia, North Italy, Austria and Germany, France, Spain and Portugal). The markers were found to be highly informative in all cultivar groups and therefore constitute a useful set for the genetic characterization of European grapevines. Similar and high levels of genetic variability were detected in all investigated grapevine gene pools. Genetic differentiation among cultivars from different regions was significant, even in the case of adjacent groups such as the Spanish and Portuguese cultivars. No genetic differentiation could be detected between vines with blue and white grapes, indicating that they have undergone the processes of cultivar development jointly. The observed genetic differentiation among vine-growing regions suggested that cultivars could possibly be assigned to their regions of origin according to their genotypes. This might allow one to determine the geographical origin of cultivars with an unknown background. The assignment procedure proved to work for cultivars from the higher differentiated regions, as for example from Austria and Portugal. Received: 10 April 1999 / Accepted: 25 May 1999     

Analysis of genetic diversity and relationships in East African banana germplasm

The genetic diversity and phylogenetic relationships of 29 East African highland banana (Musa spp.) cultivars and two outgroup taxa, M. acuminata Calcutta 4 and Agbagba were surveyed by RAPD analysis. A genetic similarity matrix was established based on the presence or absence of polymorphic amplified fragments. Phylogenetic relationships were determined by UPGMA cluster analysis. RAPDs showed that the highland bananas are closely related with a narrow genetic base. Nevertheless, there were sufficient RAPD polymorphisms that were collectively useful in distinguishing the cultivars. The dendrogram was divisible into a major cluster composed of all the AAA highland banana cultivars and Agbagba (AAB) and a minor cluster consisting of Kisubi (AB), Kamaramasenge (AB) and Calcutta 4 (AA). Several subgroups are recognized within the major cluster. RAPD data did not separate beer and cooking banana cultivars. Our study showed that RAPD markers can readily dissect genetic differences between the closely related highland bananas and provide a basis for the selection of parents for improvement of this germplasm. Received: 28 June 2000 / Accepted: 1 August 2000     

Chimerism in grapevines: implications for cultivar identity,ancestry and genetic improvement

In the course of DNA profiling of grapevine cultivars using microsatellite loci we have occasionally observed more than two alleles at a locus in some individuals and have identified periclinal chimerism as the source of such anomalies. This phenomenon in long-lived clonally propagated crops, such as grapevine, which contains historically ancient cultivars, may have a role in clonal differences and affect cultivar identification and pedigree analysis. Here we show that when the two cell layers of a periclinal chimera, Pinot Meunier, are separated by passage through somatic embryogenesis the regenerated plants not only have distinct DNA profiles which are different from those of the parent plant but also have novel phenotypes. Recovery of these phenotypes indicates that additional genetic differences can exist between the two cell layers and that the Pinot Meunier phenotype is due to the interaction of genetically distinct cell layers. It appears that grapevine chimerism can not only modify phenotype but can also impact on grapevine improvement as both genetic transformation and conventional breeding strategies separate mutations in the L1 and L2 cell layers. Received: 14 March 2001 / Accepted: 22 May 2001     

Using microsatellites,isozymes and AFLPs to evaluate genetic diversity and redundancy in the cassava core collection and to assess the usefulness of DNA-based markers to maintain germplasm collections

The cassava core collection was selected to represent, with minimum repetitiveness, the potential genetic diversity of the crop. The core (630 accessions) was chosen from the base collection (over 5500 accessions) on the basis of diversity of origin (country and geographic), morphology, isozyme patterns and specific agronomic criteria. To asses the genetic diversity of the core, 521 accessions were typed with four microsatellite loci. Allele diversity and frequency, and size variance of dinucleotide repeats (Rst statistic) were estimated. Microsatellite allele numbers and frequencies varied among countries: Colombia and Brazil had the largest number of different alleles across all loci. Mexico also had a high number, ranking fifth after Peru, Costa Rica and Venezuela (which tied). Unique alleles were present in accessions from Brazil, Colombia, Guatemala, Venezuela and Paraguay. A small number (1.34%) of potential duplicates were identified through isozyme and AFLP profiles. Thus, the present results indicated that traditional markers have been highly effective at selecting unique genotypes for the core. Future selections of cassava germplasm sets can be aided by DNA-based markers to ensure genetically representative, non-redundant samples. This revised version was published online in June 2006 with corrections to the Cover Date.     

The use of microsatellite markers for detection of genetic diversity in barley populations

 A barley lambda-phage library was screened with (GA)n and (GT)n probes for developing microsatellite markers. The number of repeats ranged from 2 to 58 for GA and from 2 to 24 for GT. Fifteen selected microsatellite markers were highly polymorphic for barley. These microsatellite markers were used to estimate the genetic diversity among 163 barley genotypes chosen from the collection of the IPK Genebank, Germany. A total of 130 alleles were detected by 15 barley microsatellite markers. The number of alleles per microsatellite marker varied from 5 to 15. On average 8.6 alleles per locus were observed. Except for GMS004 all other barley microsatellite markers showed on average a high value of gene diversity ranging from 0.64 to 0.88. The mean value of gene diversity in the wild forms and landraces was 0.74, and even among the cultivars the gene diversity ranged from 0.30 to 0.86 with a mean of 0.72. No significant differences in polymorphism were detected by the GA and GT microsatellite markers. The estimated genetic distances revealed by the microsatellite markers were, on average , 0.75 for the wild forms, 0.72 for landraces and 0.70 among cultivars. The microsatellite markers were able to distinguish between different barley genotypes. The high degree of polymorphisms of microsatellite markers allows a rapid and efficient identification of barley genotypes. Received: 26 November 1997 / Accepted: 19 January 1998     

Marker-assisted rationalisation of genetic resource collections: a case study in flax using AFLPs

Removing redundant germplasm from collections is one of the options for genebanks to increase the efficiency of their genetic resource management. Molecular characterisation of germplasm is thereby becoming more and more important to verify suspected duplication. AFLPs were used to characterise 29 flax accessions of material derived from research activities (hereafter termed ’’breeder’s line”). Based on similar accession names, the breeder’s lines could be classified into three series (’M 25’, ’Ru’ and ’Rm’) that were expected to contain redundancies. In addition, 12 reference cultivars were analysed. A total number of 144 polymorphic bands (59.8%) were scored among the 164 individuals investigated. In general, relatively high levels of intra-accession variation were found, even for the cultivars examined. This finding was not in line with the low outcrossing rates reported for flax. A cluster analysis grouped the ’Ru’ and ’Rm’ series together, indicating their close genetic relationship. An analysis of molecular variance (AMOVA) showed a significant group effect (fibre/oil flax) only for ’M 25’, explaining 34% of the variation observed within this series. For the cultivars 40.5% of the variation was distributed among accessions within groups and all pairwise comparisons were significantly different, except for one case. Both for the series of breeder’s lines and the cultivars the major part of the variation was distributed among individuals within accessions. This component constituted 80.7% and 83.6% of the total variation for the ’Ru’ and ’Rm’ series, respectively. Pairwise comparisons of accessions were performed by AMOVA in order to identify redundant germplasm. Stepwise bulking of accessions until all remaining accessions were significantly different showed that the 29 accessions of breeder’s lines could be reduced to 14. Only a small negative effect of this bulking approach on the among-population component of variance was observed, showing a reduction of 2.6%. Results are discussed in relation to improving the efficiency of collection management. Received: 25 June 2000 / Accepted: 27 October 2000     

Controlling genetic variability by mathematical programming in a selection scheme on an open-pollinated population in Eucalyptus globulus

A model using integer quadratic mathematical programming has been developed to control the inbreeding level (or genetic diversity) through group coancestry in a selection programme for a forestry population structured in terms of maternal families coming from different locations. A method to calculate the average group coancestry between- and within-families for these open-pollinated populations is also proposed. This model has been applied to data from a breeding programme of Australian Eucalyptus globulus. The strategy proved to be effective as reductions of up to 50% for the group coancestry of the selected individuals were reached with a loss of only 5% of the maximum attainable selection differential (corresponding to truncation selection). Received: 14 October 1999 / Accepted: 26 July 2000     

The use of microsatellites for detecting DNA polymorphism, genotype identification and genetic diversity in wheat

A set of 20 wheat microsatellite markers was used with 55 elite wheat genotypes to examine their utility (1) in detecting DNA polymorphism, (2)in the identifying genotypes and (3) in estimating genetic diversity among wheat genotypes. The 55 elite genotypes of wheat used in this study originated in 29 countries representing six continents. A total of 155 alleles were detected at 21 loci using the above microsatellite primer pairs (only 1 primer amplified 2 loci; all other primers amplified 1 locus each). Of the 20 primers amplifying 21 loci, 17 primers and their corresponding 18 loci were assigned to 13 different chromosomes (6 chromosomes of the A genome, 5 chromosomes of the B genome and 2 chromosomes of the D genome). The number of alleles per locus ranged from 1 to 13, with an average of 7.4 alleles per locus. The values of average polymorphic information content (PIC) and the marker index (MI) for these markers were estimated to be 0.71 and 0.70, respectively. The (GT)_n microsatellites were found to be the most polymorphic. The genetic similarity (GS) coefficient for all possible 1485 pairs of genotypes ranged from 0.05 to 0.88 with an average of 0.23. The dendrogram, prepared on the basis of similarity matrix using the UPGMA algorithm, delineated the above genotypes into two major clusters (I and II), each with two subclusters (Ia, Ib and IIa, IIb). One of these subclusters (Ib) consisted of a solitary genotype (E3111) from Portugal, so that it was unique and diverse with respect to all other genotypes belonging to cluster I and placed in subcluster Ia. Using a set of only 12 primer pairs, we were able to distinguish a maximum of 48 of the above 55 wheat genotypes. The results demonstrate the utility of microsatellite markers for detecting polymorphism leading to genotype identification and for estimating genetic diversity. Received: 15 May 1999 / Accepted: 27 July 1999     

The genetic diversity of annual wild soybeans grown in China

Annual wild soybeans (Glycine soja), the ancestors of cultivated soybeans (G. max), are important sources of major genes for resistance to pests, diseases and environmental stresses. The study of their genetic diversity is invaluable for efficient utilization, conservation and management of germplasm collections. In this paper, the number of accessions, the variation of traits, the genetic diversity indexes (Shannon index) and the coefficient of variation were employed to study the geographical distribution of accessions, genetic diversity of characters and genetic diversity centers of annual wild soybean by statistical analysis of the database from the National Germplasm Evaluation Program of China. Most annual wild soybeans are distributed in Northeast China, and the number of accessions decreases from the Northeast to other directions in China. The genetic diversity indexes (Shannon index) were 0.49, 0.74, 0.02, 0.55, 1.45, 2.41, 1.27 and 1.89 for flower color, sootiness of seed coat, cotyledon color, pubescence color, hilum color, leaf shape, stem type and seed color, respectively. Coefficients of variation were 7.1%, 28.7%, 76.43% and 18.2% for protein content, oil content, 100-seed weight and days to maturity, respectively. Three genetic diversity centers, the Northeast, the Yellow River Valley and the Southeast Coasts of China, are proposed based on the geographical distribution of the number of accessions, genetic diversity and the multivariate variation coefficient. Based on these results and Vavilov’s theory of crop origination, two opposing possible models for the formation of the three centers are proposed, either these centers are independent of each other and the annual wild soybeans in these centers originated separately, or the Northeast center was the primary center for annual wild soybeans in China, while the Yellow River Valley center was derived from this primary center and served as the origin for the Southeast Coast center. Received: 25 June 2000 / Accepted: 18 October 2000     

Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of the genetic linkage map

Inter-simple sequence repeat (ISSR) analysis was evaluated for its usefulness in generating markers to extend the genetic linkage map of Citrus using a backcross population previously mapped with restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and isozyme markers. ISSR markers were obtained through the simple technique of PCR followed by analysis on agarose gels, using simple sequence repeat (SSR) primers. Optimization of reaction conditions was achieved for 50% of the SSR primers screened, and the primers amplified reproducible polymorphic bands in the parents and progeny of the backcross population. Mendelian segregation of the polymorphic bands was demonstrated, with an insignificant number of skewed loci. Most of the SSR primers produced dominant loci; however co-dominance was observed with loci derived from three primers. A new genetic map was produced by combining the segregation data for the ISSR markers and data for the RFLP, RAPD and isozyme markers from the previous map and creating genetic linkages among all the markers using JoinMap 2.0 mapping software. The new map has an improved distribution of markers along the linkage groups with fewer gaps, and marker order showed partial or complete conservation in the linkage groups. The incorporation of ISSR markers into the genetic linkage map demonstrates that ISSR markers are suitable for genetic mapping in Citrus. Received: 3 February 2000 / Accepted: 12 May 2000     

QTL analysis of an advanced backcross of Lycopersicon peruvianum to the cultivated tomato and comparisons with QTLs found in other wild species

 A BC₃ population previously developed from a backcross of Lycopersicon peruvianum, a wild relative of tomato, into the cultivated variety L. esculentum was analyzed for QTLs. Approximately 200 BC₄ families were scored for 35 traits in four locations worldwide. One hundred and sixty-six QTLs were detected for 29 of those traits. For more than half of those 29 traits at least 1 QTL was detected for which the presence of the wild allele was associated with an agronomically beneficial effect despite the inferior phenotype of the wild parent. Eight QTLs for fruit weight could be followed through the BC₂, BC₃, and BC₄, generations, supporting the authenticity of these QTLs. Comparisons were made between the QTLs found in this study and those found in studies involving two other wild species; the results showed that while some of these QTLs can be presumed to be allelic, most of the QTLs detected in this study are ones not previously discovered. Received: 9 April 1997 / Accepted: 20 May 1997     

Efficient search for new resistant genotypes to the citrus tristeza closterovirus in the orange subfamily Aurantioideae

 Virulent isolates of the citrus tristeza virus (CTV) are continuously arising and their spread threatens the world citrus industry. Methods for effective utilization of material conserved in germplasm banks are needed in plant improvement. Two objectives are pursued in the present paper: a search for new CTV-resistant genotypes and tests of two strategies for this search. One of these tests is based on a study of genetic relationships among genera and species of the orange subfamily and the other on scores of molecular markers known to be linked to the CTV-resistant locus. Sampled plants were graft-inoculated with a mild CTV isolate (T-346) and two virulent ones (T-388 and T-305). Susceptible plants were those where CTV multiplication was detected beyond 4 months after inoculation. All cultivars of Poncirus trifoliata tested, as well as Severinia buxifolia and Atalantia ceylanica, were resistant to the three CTV isolates; Fortunella crassifolia (Meiwa kumquat) resists two of them. The finding of CTV resistance in this species, closely related to cultivated Citrus species, opens a new arena for CTV-resistance improvement of oranges and mandarines by sexual hybridization. The searching strategy based on phylogenetic data has been successful, whereas the other one may be worthwhile only when the search is restricted to the species where linkage analysis is available. A good documentation system that allows quick sampling of accessions to build up core collections and where the location of new and useful genes could be easily worked out, is suggested to enhance germplasm utilization. Received: 27 April 1997 / Accepted: 5 June 1997     

The potential of ISSR-PCR primer-pair combinations for genetic linkage analysis using the seasonal flowering locus in Fragaria as a model

ISSR-PCR has been widely used for genetic distance analysis and DNA fingerprinting but has been less well utilised for mapping purposes. A key limitation lies in the small number of primer designs available to generate useful polymorphisms. In this study, the potential of paired combinations of ISSR primers is evaluated using a test cross mapping population of 168 BC₁ individuals between Fragaria vesca f. vesca and a closely related line F. vesca f. semperflorens. Ten ISSR primers and all possible pairwise combinations between them were used to generate markers potentially linked to the locus controlling seasonal flowering in F. vesca. Band profiles of individual primers were found to be highly reproducible for band position and intensity, and only minor variation was noted in band intensity (but not in position) between different constituent mixes of primer-pair combinations. Overall, ISSR primers used in isolation produced 85 markers of which only five were specific to F. vesca. None of these markers were linked to the seasonal flowering locus. In contrast, the primer-pair combina-tions yielded 493 markers, including 14 specific to F. vesca. These markers included two located within 2.2 cM of the seasonality locus. The strengths and limitations of using pairs of ISSR primers in combination for mapping and other genetic analyses are briefly explored. Received: 12 October 2000 / Accepted: 19 January 2001