Development of microsatellite markers for white spruce (Picea glauca) and related species

We report the development of 13 primer pairs that allow the unambiguous amplification of 15 microsatellite (SSR) loci in white spruce (Picea glauca). Fourteen of these loci were polymorphic in trees sampled at three geographically separated regions of western Canada. Segregation analysis carried out on these loci confirmed a Mendelian inheritance pattern for all except two, which showed significant segregation distortion. All of these primer pairs amplified SSR loci in at least one of the other Picea species tested [black spruce (P. mariana), red spruce (P. rubens), Norway spruce (P. abies), Colorado spruce (P. pungens), sitka spruce (P. sitchensis) and Engelmann spruce (P. engelmannii)]. Given the important commercial and ecological roles of these species, this set of markers will be invaluable for their management, the improvement of commercially important traits, and the study of their ecology and genetics. Received: 18 August 2000 / Accepted: 28 September 2000     

Characterization of microsatellite markers in Fagus sylvatica L. and Fagus orientalis Lipsky

Using an enrichment procedure, we cloned microsatellite repeats from European beech (Fagus sylvatica L.) and developed primers for the amplification of microsatellite markers. Six polymorphic loci were characterized which produced 3–21 alleles in 70 individuals from one Italian population, with an observed heterozygosity between 0.58 and 0.85. All six loci amplified fragments which were polymorphic in the closely related species, Fagus orientalis, also. Owing to their very high degree of variation, these markers should be very useful in gene flow studies of these species.     

Chloroplast DNA phylogeography of Fagus crenata (Fagaceae) in Japan

 CpDNA variation in Japanese beech, Fagus crenata (Fagaceae), was studied in 45 populations distributed throughout the species' range. Two cpDNA regions were sequenced: the non-coding region between the trnL (UAA) 5′exon and trnF (GAA), and the trnK region (including matK). Thirteen distinct cpDNA haplotypes were recognized and each haplotype was found to be geographically structured. Two major clades (I and II+III) were revealed in phylogenetic analyses among the haplotypes using F. sylvatica as an outgroup. The haplotypes of Clade I were distributed mainly along the Japan Sea side of the Japanese Archipelago, while those of Clade II+III occurred chiefly along the Pacific Ocean side. Consequently, the distribution of the two major cpDNA clades suggests that there were two migration routes in the history of F. crenata; one along the Japan Sea and the other along the Pacific Ocean side of the Japanese Islands. Received March 19, 2001 Accepted November 22, 2001     

Shoot dynamics and architecture of saplings in Fagus crenata across its geographical range

 Relationships between leaf or shoot size, number, and arrangement in response to light were investigated to test the hypothesis that these characteristics are linked. In order to test this hypothesis, the divergence in allometry and shoot dynamics in saplings of Japanese beech (Fagus crenata) obtained from four populations and having different leaf sizes were examined in a nursery under both full sun and shade conditions. Trees with different leaf sizes also showed large differences in canopy structure, particularly when shade-grown saplings were compared. The final leaf mass distributions of the large-leaf populations were conical or “bottom - heavy”, while those of the small-leaf populations were planar or “top - heavy”. The slope of the allometric relations between leaf mass and shoot and branch mass in small-leaved populations were steeper than those in large-leaf populations. The four populations were classified into two growth types: populations producing a few large leaf and shoot modules corresponded to “stem growth type”, and those producing many small leaf and shoot modules corresponded to “leaf growth type”. These kinds of intra-specific variation in architecture and growth of F. crenata trees may influence the structure and dynamics of forests in accordance with differences in competitive ability or sensitivity to disturbances such as windstorm. Received: 18 March 1997 / Accepted: 21 October 1997     

Characterization of highly conserved microsatellite loci in Araucaria cunninghamii and related species

 Ten microsatellite loci are described in Araucaria cunninghamii, the first reported in the Araucariaceae. Eight were tested in sections Eutacta and Bunya, which diverged more than 200 MYA, and to the sister genus Agathis. Specific amplification products within the expected size range were obtained for six to eight loci in section Eutacta (depending on species), five loci in section Bunya and three loci in Agathis. Two of the loci (CRCAc1 and CRCAc2, both GA repeats) produced specific amplification products in all taxa, with orthology confirmed by sequence analysis. The repeats were perfect in all taxa. The flanking sequences were extremely conserved, with sequence divergence of 0% to 2.0% within Araucaria species and 2.9% to 7.5% between Araucaria and Agathis. These microsatellites represent some of the most conserved microsatellite loci reported in plants. This may be due to a low evolutionary rate in Araucariaceae genome or the loci may be closely associated with highly conserved, unreported genes. Received January 14, 2002; accepted June 14, 2002 Published online: February 4, 2003 Current address: The Centre for Identification and Diagnostics, School of Life Sciences, The University of Queensland, Brisbane 4072, Australia.     

Development and characterization of microsatellite markers for Fagus crenata Blume

Microsatellite markers can yield high‐resolution genetic profiles for individual identification, and for parentage analysis, when evaluating gene dispersal in populations. Fagus crenata is an important dominant species in the cool temperate forests in Japan, and although many studies on the species have been conducted the patterns of gene dispersal via pollen and seeds are poorly understood. In order to be better informed about gene dispersal in Fagus crenata, we have developed 16 new microsatellite loci from an enriched library of genomic DNA. These 16 loci were highly variable, with 3–40 alleles per locus and an expected heterozygosity value of 0.11–0.98.     

Highly polymorphic microsatellites of rice consist of AT repeats, and a classification of closely related cultivars with these microsatellite loci

Microsatellites consisting of AT repeats are highly polymorphic in rice genomes and can be used to distinguish between even closely related japonica cultivars in Japan. Polymorphisms of 20 microsatellite loci were determined using 59 japonica cultivars, including both domestic and modern Japanese cultivars. Although the polymorphisms of these 20 microsatellite loci indicated that the Japanese cultivars were genetically quite similar, microsatellites consisting of AT repeats showed high gene diversity even among such closely related cultivars. Combinations of these hypervariable microsatellites can be employed to classify individual cultivars, since the microsatellites were stable within each cultivar. An identification system based on these highly polymorphic microsatellites could be used to maintain the purity of rice seeds by eliminating contamination. A parentage diagnosis using 17 polymorphic microsatellite loci clearly demonstrated that plants which carried desired chromosome regions had been selected in breeding programs. Thus, these hypervariable microsatellites consisting of AT repeats should promote the selection of plants which carry desired chromosomes from genetically similar parents. Backcrossing could also help to eliminate unnecessary chromosome regions with microsatellite polymorphisms at an early stage in breeding programs. Received: 8 July 1996 / Accepted: 12 July 1996     

Development of microsatellite markers for Euchresta japonica and E. formosana (Leguminosae)

We isolated 13 microsatellite loci from Euchresta japonica, an endangered shrub species that grows in warm-temperate forests in East Asia. Of these 13 loci, only one was codominant and polymorphic with five alleles. Cross-species amplification in a related species, E. formosana, detected nine of these loci, all of which were codominant and polymorphic with 2 to 9 alleles. These markers will facilitate further studies on the genetic characteristics of these two Euchresta species.     

Development and characterization of microsatellite markers in Cucumis

This study provides a set of useful SSR markers and describes their development, characterization and application for diversity studies.Sixty one Cucumis SSR markers were developed, most of them (46) from melon (Cucumis melo L.) genomic libraries. Forty of the markers (30 melon and 10 cucumber SSRs) were evaluated for length polymorphism in a sample of 13 melon genotypes and 11 cucumber (Cucumis sativus L.) genotypes. PCR-amplification revealed up to six size alleles among the melon genotypes and up to five alleles among the cucumber genotypes, with mean gene-diversity values of 0.52 and 0.28 for melon and cucumber, respectively. These differences are in accordance with the known narrower genetic background of the cucumber. SSR data were applied to phylogenetic analysis among the melon and cucumber genotypes. A clear distinction between the ’exotic’ groups and the sweet cultivated groups was demonstrated in melon. In cucumber, separation between the two sub-species, C.sativus var. sativus and C.sativus var. hardwickii,was obtained. Conservation of SSR loci between melon and cucumber was proven by sequence comparisons. Received: 17 April 2000 / Accepted: 16 May 2000     

Development and characterization of microsatellite markers in black poplar (Populus nigra L.)

Using an enrichment procedure, we have cloned and sequenced microsatellite loci from black poplar (Populus nigra L.) and developed primers for sequence-tagged microsatellite (STMS) analysis. Twelve primer pairs for dinucleotide repeats produced fragments of sufficient quality which were polymorphic in P. nigra. Some of them also showed amplification in other Populus species (P. deltoides, P. tricocarpa, P. tremula, P. tremuloides, P. candicans, and/or P. lasiocarpa). The best nine and (GT) (GA) microsatellite markers were tested on a set of 23 P. nigra genotypes from all over Europe. The microsatellites were highly polymorphic, with 10–19 different alleles per microsatellite locus among these 23 genotypes. WPMS08 sometimes amplified three fragments. Using the other eight marker loci, the level of heterozygosity among the plants was on average 0.71 (range 0.25–1.00). The microsatellite markers developed will be useful for screening the genetic diversity in natural populations and in gene bank collections. Received: 21 October 1999 / Accepted: 24 November 1999     

Species boundaries and interrelationships of two closely related sympatric diploid wild potato species, Solanum astleyi and S. boliviense, based on RAPDs

 The more than 200 wild and cultivated species relatives of potato (Solanum sect. Petota) present a valuable germplasm base for cultivar improvement. However, species boundaries and interrelationships within sect. Petota are controversial, inhibiting the efficient organization of the many germplasm collections of these species. One controversy involves questions of species boundaries and interrelationships of S. astleyi and S. boliviense. Solanum boliviense is narrowly endemic to two Departments in southern Bolivia, and S. astleyi is known only from one site entirely within the range of this species, where they co-occur. Both species are diploid and morphologically very similar. Artificial hybrids between them are fully fertile, and the species putatively hybridize naturally. These data have been interpreted to designate them as separate species or as S. astleyi an ecotype of S. boliviense. Putative progenitors of S. astleyi are S. boliviense, S. megistacrolobum subsp. megistacrolobum, and S. megistacrolobum subsp. toralapanum. We evaluated interrelationships among these species with random amplified polymorphic DNA’s (RAPDs) generated for 2 accessions of S. astleyi and 14 accessions of S. boliviense. These represent the entire geographic range of the former species and nearly the entire range of the latter. We also analyzed 1 accession each of S. acaule subsp. acaule, S. acaule subsp. aemulans, S. albicans, S. berthaultii, S. megistacrolobum subsp. megistacrolobum, S. megistacrolobum subsp. toralapanum, S. raphanifolium, S. sogarandinum, and S. sparsipilum. Phenetic analyses of the RAPD data show S. astleyi and S. boliviense to form two distinct groups and to be more similar to each other than to any of the other species investigated, suggesting that S. astleyi and S. boliviense are sister taxa. The divergence of S. astleyi and S. boliviense relative to other species examined suggests that they are worthy of taxonomic recognition at the subspecies, rather than species level, and we propose the new combination S. boliviense subsp. astleyi. Received: 21 November 1996 / Accepted: 18 April 1997     

Development, characterization and mapping of microsatellite markers in Eucalyptus grandis and E. urophylla

 We report on the development, genetic characterization and linkage mapping of a battery of SSR (simple sequence repeat) loci in Eucalyptus grandis and E. urophylla. This study reveals the abundance of SSRs in Eucalyptus, the very high information content of these markers for mapping and individual identification, and demonstrates the feasibility of constructing a comprehensive microsatellite-based linkage map for Eucalyptus. Primer sequence for a set of 20 highly informative EMBRA (Eucalyptus microsatellites from Brazil) loci are made available together with their map position and estimates of the expected heterozygosity and allele size range in these two species. Using genomic library enrichment and anchored-PCR screening prior to sequencing, the efficiency of SSR marker locus development was 63% from sequencing data to operationally useful SSR loci. Absolute transportability between the two species and very high levels of allelic variability and expected heterozygosity (H) were seen at all SSR loci surveyed. The number of alleles per locus ranged from 9 to 26 with an average of 16.3±4.8. The average H of 15 loci was 0.86±0.04, 0.83±0.08 and 0.89±0.04, respectively, for E. urophylla, E. grandis and the combined two-species estimate. In the mapping analysis 16 out of 20 marker loci segregated in a fully informative configuration, allowing the determination of synteny of six homologous linkage groups between the two species. The availability of transportable, multiallelic, PCR-based co-dominant SSR loci represents a dramatic improvement in our ability to carry out detailed population genetic analysis and to search, understand, and manipulate allelic variation at QTLs (quantitative trait loci) in species of Eucalyptus. Received: 16 March 1998 / Accepted: 22 March 1998     

Development of microsatellite markers in white birch (Betula platyphylla var. japonica)

Betula platyphylla var. japonica is a typical pioneer tree species in the secondary succession in northern Japan. We describe the cloning and characterization of 13 polymorphic, codominant microsatellite loci isolated from this species. These polymorphic loci had 2–8 alleles per locus and a range of expected heterozygosities from 0.050 to 0.808.     

Optimization of the choice of molecular markers for varietal identification in Vitis vinifera L.

 The aim of this study was to develop a cultivar identification tool based on molecular analysis and a statistical approach. From the PIC parameter we defined the D parameter, which evaluates the efficiency of a primer for the purpose of identification of varieties; i.e. the probability that two randomly chosen individuals have different patterns. D can be used to compare different types of markers even if only the allelic frequencies are known. We used this parameter to develop an algorithm for selecting the optimal combination of primers necessary to identify a set of varieties. The optimal combination of primers determined for a small elite group of varieties applied on a larger set induces a risk of confusion involving 1 of the elite varieties. We estimated the risk of confusion using the D value of each primer of the combination. We applied this methodology on a set of 224 varieties of Vitis vinifera screened with 21 RAPD primers and two microsatellite loci. The discriminating power of the primers did not only depend on the number of patterns it generates but also on the frequencies of the different patterns. A combination of 8 primers (6 RAPD and two microsatellite) was found to be optimum for the discrimination of these 224 varieties. A subset of 38 elite varieties was also investigated. The determined optimal combination of 4 primers (3 RAPD and one microsatellite) applied on the 224 varieties gave 9 risks of confusion involving 1 of the elite varieties. Confusion can happen between varieties with the same origin as well as between varieties of very diverse geographical origins. Received: 22 April 1998 / Accepted: 9 June 1998     

Erysiphe wadae: a new species of Erysiphe sect. Uncinula on Japanese beech

 A new species of Erysiphe sect. Uncinula is described and illustrated from Japan. Erysiphe wadae sp. nov., found on Japanese beech (Fagus crenata, Fagaceae), is characterized by having two types of appendages, i.e., a long (true) appendage arising from the equatorial zone of the ascomata, and a short appendage arising from the upper part of the ascomata. This characteristic is shared by E. simulans, E. australiana, E. flexuosa, E. liquidambaris, E. prunastri, and E. togashiana. Erysiphe wadae differs from the latter five species in its brown-colored appendage. Erysiphe simulans is most similar to E. wadae, but differs in its loosely uncinate appendage and smaller number of ascospores. Identity of the nucleotide sequences of the rDNA ITS region is 92.3% between the two species. The significance of the two types of appendage in taxonomy and phylogeny of powdery mildews is discussed based on molecular phylogenetic analysis. Received: November 8, 2002 / Accepted: January 29, 2003 Acknowledgments We are grateful to Drs. Yukio Harada and Hideki Naito for help in collecting powdery mildew specimens; Dr. Uwe Braun for providing the specimen of E. flexuosa; and Mr. Tetsuya Hirata and Miss Sanae Matsuda for nucleotide sequences of E. togashiana and E. flexuosa. This work was partially supported by a Grant-in-Aid for Scientific Research (No. 13660047) from Ministry of Education, Culture, Sports, Science and Technology, Japan.     

Assessment of genetic relationships and population discrimination among Fagus sylvatica L. by RAPD

 We assessed the genetic relationships between members of the Fagaceae family by RAPDs in order to better ascertain the taxonomic status of a very particular population of Fagus sylvatica, the ‘tortuosa’ variety. Intra- and inter-population Nei and Li’s mean genetic distances were compared, and the genetic relationships between individuals were clarified on dendrograms by the Neighbor joining method. RAPD analysis was first conducted on three species from three genera, Quercus petraea, Castanea sativa, and Fagus sylvatica, in order to develop an efficient RAPD protocol. The variety level was then studied, and a general tendency of the individuals to cluster by variety was observed. Individuals also clustered by geographic locations, but the genetic distances between populations were not correlated to the distances between sites. Finally, we compared the common beech and ‘tortuosa’ varieties from two different locations, Verzy and Süntel. Both populations from one location were closer than the same variety from two sites. This last result is in agreement with those previously obtained with isozymes. Hypotheses concerning the origin of the ‘tortuosa’ variety are discussed. Received: 9 January 1998 / Accepted: 25 February 1998     

Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers

 Inter-simple sequence repeat polymorphic DNA (ISSR) was evaluated for its applicability as a genetic marker system in wheat. PCR was carried out with primers that annealed to simple sequence repeats. The resultant products were subjected to agarose-gel electrophoresis, and the banding patterns were compared among six wheat accessions containing diploid, tetraploid, and hexaploid members. Out of 100 examined, 33 primers produced distinguishable as well as polymorphic bands in each of the six accessions. Although most of the primers that gave distinct bands (30 primers out of 33) contained dinucleotide repeats, each of the primers with tri-, tetra-, and penta-nucleotide motifs also yielded discrete bands. Primers based on (AC)_n repeats gave the most polymorphic bands. In total, 224 polymorphic bands were found in the comparison between Einkorn wheats whereas, on the average, 120 polymorphic bands were detected between common wheats. ISSR primers produced several times more information than RAPD markers. The extent of band polymorphism was similar to that of RFLP markers, and greater than that of RAPDs. The genetic relationships of wheat accessions estimated by the polymorphism of ISSR markers were identical with those inferred by RFLP and RAPD markers, indicating the reliability of ISSR markers for estimation of genotypes. These polymorphic bands are potential candidates as novel markers for use in linkage-map construction in wheat. The characteristic features of ISSR markers, i.e. polymorphism, generation of information and ease of handling, suggest their applicability to the analysis of genotypes as well as to the construction of PCR-based genome maps of wheats. Received: 15 September 1996 / Accepted: 25 October 1996     

Characterisation and analysis of microsatellite loci in a mangrove species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

An enriched microsatellite library of the mangrove species Avicennia marina was constructed, in which 85.8% of the clones contained microsatellite sequences. Of the microsatellite repeat sequences isolated, 55.0% were di-nucleotides, 34.2% were tri-nucleotides, 50.0% were perfect, 24.2% were imperfect, and 15.0% were compound. Four different di-nucleotide repeats were isolated with repeat lengths ranging from 5 to 33; ten different tri-nucleotide repeats were isolated with repeat lengths ranging from 3 to 25. The most common di-nucleotide was the AC/TG repeat; the most common tri-nucleotide was the CCG/GGC repeat. Sixteen microsatellite sequences were selected for primer design, and 6 primers were selected to investigate the polymorphism detected among 15 individuals of A. marina from three natural populations in Australia. A total of 40 alleles were detected at 6 microsatellite loci. The number of alleles per microsatellite locus ranged from 5 to 13. On average, 7 alleles were detected per locus. All microsatellite loci showed high levels of gene diversity (heterozygosity), with values ranging from 0.53 to 0.88; the mean value of gene diversity was 0.70. Microsatellite loci were also tested for conservation across Avicennia species. There was a decline in amplification success with increasing divergence between Avicennia species. The results indicate that microsatellites are abundant in the Avicennia genome and can be valuable genetic markers for assessing the effects of deforestation and forest fragmentation in mangrove communities, which is an important issue for mangrove conservation and afforestation schemes. Received: 8 June 1999 / Accepted: 21 September 1999     

The development of oat microsatellite markers and their use in identifying relationships among Avena species and oat cultivars

Microsatellites have many desirable marker properties. There has been no report of the development and utilization of microsatellite markers in oat. The objectives of the present study were to construct oat microsatellite-enriched libraries, to isolate microsatellite sequences and evaluate their level of polymorphism in Avena species and oat cultivars. One hundred clones were isolated and sequenced from three oat microsatellite-libraries enriched for either (AC/TG) _n , (AG/TC) _n or (AAG/TTC) _n repeats. Seventy eight clones contained microsatellites. A database search showed that 42% of the microsatellite flanking sequences shared significant homology with various repetitive elements. Alu and retrotransposon sequences were the two largest groups associated with the microsatellites. Forty four primer sets were used to amplify the DNA from 12 Avena species and 20 Avena sativa cultivars. Sixty two percent of the primers revealed polymorphism among the Avena species, but only 36% among the cultivars. In the cultivars, the microsatellites associated with repetitive elements were less polymorphic than those not associated with repetitive elements. Only 25% of the microsatellites associated with repetitive elements were polymorphic, while 46% of the microsatellites not associated with repetitive elements showed polymorphism in the cultivars. An average of four alleles with a polymorphism information content (PIC) of 0.57 per primer set was detected among the Avena species, and 3.8 alleles with a PIC of 0.55 among the cultivars. In addition, 54 barley microsatellite primers were tested in Avena species and 26% of the primers amplified microsatellites from oat. Using microsatellite polymorphisms, dendrograms were constructed showing phylogenetic relationships among Avena species and genetic relationships among oat cultivars. Received: 1 November 1999 / Accepted: 14 April 2000