Molecular characterization and similarity relationships among apricot ( Prunus armeniaca L.) genotypes using simple sequence repeats

A collection of 48 apricot genotypes, originated from diverse geographic areas, have been screened with 37 SSR primer pairs developed in different species of Prunus in order to identify and characterize the genotypes and establish their genetic relations. Thirty one of those primer pairs resulted in correct amplifications and 20 produced polymorphic repeatable amplification patterns with the 48 genotypes studied. A total of 82 alleles were detected for the 20 loci. All the genotypes studied could be unequivocally distinguished with the combination of SSRs used. The results obtained evidence for the cross-species transportability of microsatellite sequences, allowing the discrimination among different genotypes of a given fruit-tree species with sequences developed in other species. UPGMA cluster analysis of the similarity data grouped the genotypes studied according to their geographic origin and/or their pedigree information. Received: 5 April 2001 / Accepted: 4 May 2001     

Isolation and characterization of polymorphic microsatellites in olive (Olea europaea L.) and their transferability to other genera in the Oleaceae

Seven polymorphic microsatellites were developed in olive. Six of them came from a genomic library enriched for GA and CA repeat sequences. They showed single locus polymorphism in a set of 23 olive cultivars (from six to nine alleles per locus). Three different pairs of loci were sufficient to discriminate all cultivars. The other polymorphic primer pair was designed from a published sequence for olive lupeol sgutase and revealed just two alleles. The seven primer pairs were tested on two accessions of five other species of the Oleaceae and three, EMO2, EMO13 and EMO90, revealed polymorphism in two, four and three species, respectively.     

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     

Genomic and cDNA microsatellites from apricot (Prunus armeniaca L.)

We developed primers for the amplification of 24 polymorphic nuclear microsatellites in apricot (Prunus armeniaca L.). Thirteen loci originated from three genomic libraries enriched for TC, TG and AAG motifs. Eight loci were developed from three fruit EST (Expressed‐Sequence‐Tag) libraries and three from a leaf cDNA microsatellite‐enriched library. There were up to nine alleles per polymorphic locus in 12 different cultivars. No difference in allele numbers were shown between cDNA and genomic‐source loci. Mean expected heterozygosity was 0.65 (range: 0.15–0.87). Mendelian segregation was confirmed for all loci. These markers should be helpful for diversity studies, genome mapping and cultivar identification in apricot and related species.     

Isolation and characterization of polymorphic microsatellite markers from European pear (Pyrus communis L.)

This study reports the development and characterization of 19 microsatellite primer pairs developed from genomic DNA of European pear (Pyrus communis) and their transferability to other Pyrus and Malus material. The primers were designed from two different genomic libraries enriched for di‐ and trinucleotide repeats. When tested in six P. communis cultivars and 15 other Pyrus species, 13 primers revealed single‐locus polymorphism and six showed more complex patterns that suggest multiple loci. Two to 18 alleles were detected per locus and two primer pairs were sufficient to discriminate all accessions. Transferability of nine primer pairs to Malus was demonstrated through amplification of discrete products in two accessions.     

Isolation and characterization of microsatellites in Brassica rapa L

We report here the isolation and characterization of microsatellites, or simple sequence repeats (SSRs), in Brassica rapa. The size-fractionated genomic library was screened with (GA)(15) and (GT)(15) oligonucleotide probes. A total of 58 clones were identified as having the microsatellite repeats, and specific primer pairs were designed for 38 microsatellite loci. All primer pairs, except two, amplified fragments having the sizes expected from the sequences. Of the 36 primer pairs, 35 amplified polymorphic loci in 19 cultivars of B. rapa, while monomorphism was observed in only one primer pair. A total of 232 alleles was identified by the 36 primer pairs in 19 cultivars of B. rapa, and these primer pairs were examined also in nine Brassicaceae species. Most of the 36 primer pairs amplified the loci in the Brassicaceae species. Segregation of the microsatellites was studied in an F(2) population from a cross of doubled-haploid lines DH27 x G309. The microsatellites segregated in a co-dominant manner. These results indicate that the microsatellites isolated in this study were highly informative and could be useful tools for genetic analysis in B. rapa and other related species.     

Trinucleotide repeat microsatellite markers for black poplar (Populus nigra L.)

Using an enrichment procedure, we have cloned microsatellite repeats from black poplar (Populus nigra L.) and developed primers for microsatellite marker analysis. Ten primer pairs, mostly for trinucleotide repeats, produced polymorphic fragments in P. nigra. Some of them also showed amplification in other poplar species. (P. deltoides, P. tricocarpa, P. tremula, P. tremuloides, P. candicans, P. lasiocarpa). The best six loci were tested on 23 P. nigra genotypes collected across Europe. The microsatellites produced up to 12 alleles per locus in this set, with observed heterozygosity between 0.32 and 0.91.     

Eleven new microsatellites for hop (Humulus lupulus L.)

We present a new set of 11 polymorphic microsatellite primer sequences for use with Humulus lupulus. Microsatellite‐enriched libraries for GA_n and GT_n types of repeats were produced. Sequencing of 72 clones revealed 42 unique inserts containing microsatellites, out of which 19 primer pairs were designed and microsatellite amplification was tested on 39 wild hops and cultivars. Eleven primer pairs showed single locus amplification with 2–13 alleles, average 7.2, of which 17 unique alleles were discovered. One primer pair amplified too strong stutter bands, one locus was monomorphic and multilocus amplification was obtained with the remaining six primer pairs.     

Characterization of microsatellites in wild and sweet cherry (Prunus avium L.)--markers for individual identification and reproductive processes.

Nuclear microsatellites were characterized in Prunus avium and validated as markers for individual and cultivar identification, as well as for studies of pollen- and seed-mediated gene flow. We used 20 primer pairs from a simple sequence repeat (SSR) library of Prunus persica and identified 7 loci harboring polymorphic microsatellite sequences in P. avium. In a natural population of 75 wild cherry trees, the number of alleles per locus ranged from 4 to 9 and expected heterozygosity from 0.39 to 0.77. The variability of the SSR markers allowed an unambiguous identification of individual trees and potential root suckers. Additionally, we analyzed 13 sweet cherry cultivars and differentiated 12 of them. An exclusion probability of 0.984 was calculated, which indicates that the seven loci are suitable markers for paternity analysis. The woody endocarp was successfully used for resolution of all microsatellite loci and exhibited the same multilocus genotype as the mother tree, as shown in a single seed progeny. Hence, SSR fingerprinting of the purely maternal endocarp was also successful in this Prunus species, allowing the identification of the mother tree of the dispersed seeds. The linkage of microsatellite loci with PCR-amplified alleles of the self-incompatibility locus was tested in two full-sib families of sweet cherry cultivars. From low recombination frequencies, we inferred that two loci are linked with the S locus. The present study provides markers that will significantly facilitate studies of spatial genetic variation and gene flow in wild cherry, as well as breeding programs in sweet cherry.     

Isolation and characterization of microsatellite markers for red elm (Ulmus rubra Muhl.) and cross-species amplification with Siberian elm (Ulmus pumila L.)

Ulmus pumila is an elm species, non-native to the USA that hybridizes with Ulmus rubra. In order to study the genetic structure and hybridization patterns between these two elm species, we developed 15 primer pairs for microsatellite loci in U. rubra and tested their cross-amplification in U. pumila. All 15 primers amplified in both species, 11 of which possessed species-specific alleles. Eight loci were polymorphic in U. pumila and eight in U. rubra, each with two to eight alleles per locus. In addition, five primer pairs previously developed in U. laevis and U. carpinifolia (syn. U. minor) cross-amplified and showed polymorphic loci in U. pumila and/or U. rubra. These markers will facilitate the study of genetic structure and gene flow between U. rubra and exotic, invasive U. pumila.     

Isolation,characterization and cross‐species amplification of microsatellite DNA loci in the tropical American yam Dioscorea trifida

We developed microsatellite markers in American yam (Dioscorea trifida). A microsatellite sequence‐enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Among these, eight primer pairs yielded amplification products that were both interpretable and polymorphic in 24 yam cultivars. The number of alleles per locus ranged from two to 13 and the overall expected heterozygosity was around 0.5. Six of the eight Dioscorea trifida microsatellite loci gave amplification products in other Dioscorea species.     

Identification of polymorphic microsatellite markers from RAPD product in turbot (Scophthalmus maximus) and a test of cross‐species amplification

Five polymorphic microsatellite loci have been isolated and characterized from random amplified polymorphic DNA product in turbot, Scophthalmus maximus. Twelve microsatellites were selected for designing microsatellite primers, of which five gave working primer pairs. They had between four and nine alleles. Observed and expected heterozygosities varied from 0.76 to 0.90 and from 0.63 to 0.83, respectively. Five additional fish species assessed for cross‐species amplification revealed between one and three positive amplifications and between zero and two polymorphic loci per species.     

Isolation and characterization of microsatellite loci from yellowtail Seriola quinqueradiata and cross‐species amplification within the genus Seriola

We developed five microsatellite primer pairs for the yellowtail Seriola quinqueradiata. The loci were highly polymorphic, with eight to 14 alleles per locus, and can be used to study kinship and/or population structure. Many of these primer pairs amplified polymorphic loci in cross‐species amplification tests for two other Seriola species (S. lalandi and S. dumerili).     

Development of 25 gene‐associated microsatellite markers of Atlantic cod (Gadus morhua L.)

Microsatellites were identified by screening 2294 GenBank entries available for Atlantic cod (Gadus morhua L.), mainly representing expressed sequence tags and cDNA sequences. Ninety‐two novel microsatellite loci (tetra‐, tri‐ and dinucleotides) were characterized on 96 individuals. This strategy yielded 25 gene‐associated polymorphic microsatellite markers (11 tri‐ and 14 dinucleotides) with two to 20 alleles and an average heterozygosity of 0.48 in the population studied (range 0.02–0.89). One marker exhibited significant homozygote excess, and one of the primer pairs amplified two linked markers. The gene identity was determined at nine of the loci, confirming the associated microsatellites as type I markers.     

Microsatellite markers for Spergularia media (L.) C. Presl. (Caryophyllaceae) and their cross-species transferability

We developed 11 polymorphic microsatellite markers for Spergularia media (Caryophyllaceae), a perennial halophyte of coastal salt meadows and continental areas of western Eurasia. The number of alleles per locus observed in a single population of 20 individuals from the German North Sea coast ranged from 3 to 20. Observed and expected heterozygosities ranged from 0.200 to 0.850 and from 0.278 to 0.936 respectively. Observed heterozygosities were lower than expected heterozygosities at all loci, presumably as a consequence of inbreeding. All markers cross-amplified in the closely related S. salina. Of these, nine were polymorphic in the heterologous species. Only two primer pairs generated PCR products in S. diandra.     

Development and characterization of polymorphic microsatellites from Prunus avium‘Napoleon’

Primers were developed for 21 microsatellite loci isolated by enrichment from Prunus avium‘Napoleon’. Twelve loci contained uninterrupted dinucleotide repeats and nine were more complex. Nineteen primer pairs (EMPA001–019) showed single locus polymorphisms in a cultivar survey of 14 sweet cherries, with two to seven alleles per locus. Three primer pairs in combination (EMPA014, 015 and 018) discriminated all cultivars. Two primer pairs for loci monomorphic in P. avium were included: EMPA020 revealed segregation in an interspecific progeny and EMPA021 revealed polymorphism in P. dulcis. Twelve primer pairs reliably amplified products in three peach cultivars of which seven revealed polymorphisms.     

Development of microsatellite markers in peach [ Prunus persica (L.) Batsch] and their use in genetic diversity analysis in peach and sweet cherry ( Prunus avium L.)

We report the sequence of 41 primer pairs of microsatellites from a CT-enriched genomic library of the peach cultivar 'Merrill O'Henry'. Ten microsatellite-containing clones had sequences similar to plant coding sequences in databases and could be used as markers for known functions. For microsatellites segregating at least in one of the two Prunus F(2) progenies analyzed, it was possible to demonstrate Mendelian inheritance. Microsatellite polymorphism was evaluated in 27 peach and 21 sweet cherry cultivars. All primer pairs gave PCR-amplification products on peach and 33 on cherry (80.5%). Six PCR-amplifications revealed several loci (14.6%) in peach and eight (19.5%) in sweet cherry. Among the 33 single-locus microsatellites amplified in peach and sweet cherry, 13 revealed polymorphism both in peach and cherry, 19 were polymorphic only on peach and one was polymorphic only on cherry. The number of alleles per locus ranged from 1 to 9 for peach and from 1 to 6 on sweet cherry with an average of 4.2 and 2.8 in peach and sweet cherry, respectively. Cross-species amplification was tested within the Prunus species: Prunus avium L. (sweet cherry and mazzard), Prunus cerasus L. (sour cherry), Prunus domestica L. (European plum), Prunus amygdalus Batsch. (almond), Prunus armeniaca L. (apricot), Prunus cerasifera Ehrh. (Myrobalan plum). Plants from other genera of the Rosaceae were also tested: Malus (apple) and Fragaria (strawberry), as well as species not belonging to the Rosaceae: Castanea (chestnut tree), Juglans (walnut tree) and Vitis (grapevine). Six microsatellites gave amplification on all the tested species. Among them, one had an amplified region homologous to sequences encoding a MADS-box protein in Malus x domestica. Twelve microsatellites (29.3%) were amplified in all the Rosaceae species tested and 31 (75.6%) were amplified in all the six Prunus species tested. Thirty three (80.5%), 18 (43.9%) and 13 (31.7%) gave amplification on chestnut tree, grapevine and walnut tree, respectively.     

Isolation of microsatellite loci in the pollinating fig wasp of Ficus hispida, Ceratosolen solmsi

Microsatellite loci were isolated for Ceratosolen solmsi , pollinator of the dioecious Ficus hispida. We developed nine polymorphic microsatellite loci based on the method of polymerase chain reaction isolation of microsatellite arrays (PIMA). Enrichment of genomic libraries was performed by random amplified polymorphic DNA (RAPD). A subset of 38 positive clones was sequenced; 15 clones showed microsatellite loci. We tested 15 designed primer pairs and nine of them produced polymorphic amplification in 48 individual wasps collected from different fruits of the dioecious host fig Ficus hispida in China. Among the 48 individuals, 49 alleles were obtained at the nine loci. The observed heterozygosity ranged between 0.357 and 0.634.     

Development, characterization and inheritance of new microsatellites in olive (Olea europaea L.) and evaluation of their usefulness in cultivar identification and genetic relationship studies

Twelve new microsatellites have been developed in olive. For that purpose, a genomic library of the olive cultivar ‘Arbequina’ was enriched for GA, GT and ACT repeats. Two methods of screening yielded 27 sequences containing microsatellites out of the 119 clones sequenced. The GA repeat seems to be the most abundant motif. Among sequences containing microsatellites, 4 (14.8%) were redundant, 1 (3.7%) was previously described in the literature and 12 (44.4%) could not be used for primers design because the repeat motifs were incomplete. Suitable primer pairs were obtained for the remaining 10 (37.0%) sequences plus an additional 14 recovered from a formerly developed library. For the 24 primer pairs designed, 4 failed to amplify, 8 produced a complex bands pattern and 12 succeeded in giving amplification products. Considering these 12 primer pairs, 10 showed single locus amplification, whereas the other 2 revealed two loci each. This was demonstrated by studying allele segregation in two olive progenies. Sixty-eight alleles were detected for the 12 microsatellites when 51 olive cultivars were analysed. The number of alleles per locus ranged from 1 to 13. The expected heterozygosity varied between 0 and 0.83. All pairs of cultivars could be distinguished using only three microsatellites due to their great discrimination power value. The data coming from genotyping the 51 olive cultivars for 7 out of the 12 new microsatellites were used for constructing a dendrogram by unweighted pair group method with arithmetic mean cluster analysis using the Dice similarity coefficient. Cultivar association according to their geographical origin was observed.     

Development of simple sequence repeats (SSRs) in olive tree (Olea europaea L.)

We report the development of microsatellites or simple sequence repeats (SSRs) in the olive tree (Olea europaea L.). Forty three positive clones obtained by the screening of a GA-enriched genomic library were sequenced and primers were designed for 13 microsatellite loci. Five primer pairs amplified polymorphic products of the expected size range. SSR polymorphism was explored in a set of 46 olive cultivars. A total of 26 alleles were detected for the five loci. Heterozygosity ranged from 0.46 to 0.71. Ninety one per cent of the cultivars had unique multilocus genotypes. Microsatellite segregation was studied in a complex population from a cross between the commercial cultivars ’Leccino’ and ’Dolce Agogia’. Received: 3 February 2000 / Accepted: 21 March 2000