New set of microsatellite loci isolated in apricot

We report 99 simple sequence repeats (SSRs) newly isolated from an apricot (Prunus armeniaca L.) genomic library enriched for AG/CT repeats. Twenty SSRs were screened for their polymorphism in 16 apricot cultivars. The number of alleles ranged from two to nine, whereas the expected heterozygosity (H_E) ranged from 0.26 to 0.82. The same SSRs showed also an appreciable transportability across different Prunus species, such as peach, nectarine, almond, European plum, Japanese plum, sweet cherry and sour cherry, with 20% of primers giving successful amplifications in all Prunus species assayed. None gave amplification in apple.     

Microsatellites isolated in almond from an AC‐repeat enriched library

We have isolated 44 SSRs from an AC‐enriched genomic library from almond (Prunus amygdalus Batsch.). Twenty SSRs were screened for their polymorphism in 16 cultivars and for their transportability in seven different Prunus species (peach, nectarine, apricot, European plum, Japanese plum, sweet cherry, sour cherry) and in apple. The expected heterozygosity ranged from 0.62 to 0.89. About 30% of primers gave successful amplification in seven different Prunus species; in two cases amplifications were obtained also in apple.     

AC/GT and AG/CT microsatellite repeats in peach [Prunus persica (L) Batsch]: isolation, characterisation and cross-species amplification in Prunus

 We report the sequences of 17 primer pairs of microsatellite loci, which we have cloned and sequenced from two genomic libraries of peach [Prunus persica (L) Batsch] ‘Redhaven’, enriched for AC/GT and AG/CT repeats respectively. For ten of these microsatellite loci we were able to demonstrate Mendelian inheritance in a segregating back-cross population; the remainder did not segregate. The polymorphism of the microsatellites was evaluated in a panel of ten peach genotypes, including true-to-type peaches, nectarines and one canning-peach. Fifteen microsatellites (88%) were polymorphic showing 2–4 alleles each. The mean heterozygosity, averaged over all loci, was 0.32 and significantly higher than that reported in the literature for isozymes and molecular markers, such as RFLPs and RAPDs. We have also assayed the cross-species transportability and found that ten microsatellite (59%) gave apparently correct amplification in all Prunus species surveyed, namely P. domestica (European plum), P. salicina (Japanese plum), P. armeniaca (apricot), P. dulcis (almond), P. persica var. vulgaris (peach), P. persica var. laevis (nectarine), P. avium (sweet cherry) and P. cerasus (sour cherry), with three of them also being amplified in Malus (apple). The remaining microsatellites gave less-extensive amplification. Because of their appreciable polymorphism and wide cross-species transportability, most of these new markers can be integrated into the linkage maps which are currently being constructed in peach, as well as in other stone fruit crops, such as almond, apricot, cherry and plum. Received: 3 September 1998 / Accepted: 28 November 1998     

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.     

Development and transportability across Prunus species of 42 polymorphic almond microsatellites

We report 47 new simple sequence repeats (SSRs) obtained from a CT/AG enriched genomic library of almond cv. Texas (syn. Mission). Forty‐two of them were polymorphic in a sample of eight almond cultivars and 31 of these were single‐locus. The average values of the number of alleles per locus (6.6), and mean observed (65%) and expected (76%) heterozygosities for these 31 SSRs indicated a high level of variability. All cultivars studied could be individually identified using any one of the five SSRs. Transportability to other Prunus species (peach, sweet cherry, Japanese plum and apricot) was also high (83–100%).     

Ten novel microsatellite markers for the freshwater sleeper Micropercops swinhonis (Günther, 1873), with testing of cross‐species amplification in two other fishes from suborder Gobioidei

Ten novel polymorphic microsatellite loci were developed for the freshwater sleeper Micropercops swinhonis, a relatively abundant and broadly distributed lake‐dwelling fish in Asia, using next generation sequencing. These markers were tested in a population of 48 individuals, and characteristics suggest that they may be useful for population genetic studies. The average number of alleles per marker was 6.6 (range: 2–26), the average expected heterozygosity was 0.55 (range: 0.081–0.955), and the average polymorphic information content value per marker was 0.507 (range: 0.077–0.942). No marker showed significant deviation from Hardy‐Weinberg Equilibrium following Bonferroni correction of significance values, and no markers showed evidence of null alleles, large allele dropout, or scoring errors from stutter bands. Two markers had successful amplification in Odontobutis potamophila, and one marker had successful amplification in Rhinogobius giurinus. These novel markers may be useful for further research in population genetic studies involving M. swinhonis.     

Characteristics and transferability of new apple EST-derived SSRs to other Rosaceae species

Genic microsatellites or simple sequence repeat markers derived from expressed sequence tags (ESTs), referred to as EST–SSRs, are inexpensive to develop, represent transcribed genes, and often have assigned putative function. The large apple (Malus × domestica) EST database (over 300,000 sequences) provides a valuable resource for developing well-characterized DNA molecular markers. In this study, we have investigated the level of transferability of 68 apple EST–SSRs in 50 individual members of the Rosaceae family, representing three genera and 14 species. These representatives included pear (Pyrus communis), apricot (Prunus armeniaca), European plum (P. domestica), Japanese plum (P. salicina), almond (P. dulcis), peach (P. persica), sour cherry (P. cerasus), sweet cherry (P. avium), strawberry (Fragaria vesca, F. moschata, F. virginiana, F. nipponica, and F. pentaphylla), and rose (Rosa hybrida). All 68 primer pairs gave an amplification product when tested on eight apple cultivars, and for most, the genomic DNA-derived amplification product matched the expected size based on EST (in silico) data. When tested across members of the Rosaceae, 75% of these primer pairs produced amplification products. Transferability of apple EST–SSRs across the Rosaceae ranged from 25% in apricot to 59% in the closely related pear. Besides pear, the highest transferability of these apple EST–SSRs, at the genus level, was observed for strawberry and peach/almond, 49 and 38%, respectively. Three markers amplified in at least one genotype within all tested species, while eight additional markers amplified in all species, except for cherry. These 11 markers are deemed good candidates for a widely transferable Rosaceae marker set provided their level of polymorphism is adequate. Overall, these findings suggest that transferability of apple EST–SSRs across Rosaceae is varied, yet valuable, thereby providing additional markers for comparative mapping and for carrying out evolutionary studies.     

<Emphasis Type="Italic">Prunus</Emphasis> microsatellite marker transferability across rosaceous crops

A total of 145 microsatellite primer pairs from Prunus DNA sequences were studied for transferability in a set of eight cultivars from nine rosaceous species (almond, peach, apricot, Japanese plum, European plum, cherry, apple, pear, and strawberry), 25 each of almond genomic, peach genomic, peach expressed sequence tags (EST), and Japanese plum genomic, 22 of almond EST, and 23 of apricot (13 EST and 10 genomic), all known to produce single-locus and polymorphic simple-sequence repeats in the species where they were developed. Most primer pairs (83.6%) amplified bands of the expected size range in other Prunus. Transferability, i.e., the proportion of microsatellites that amplified and were polymorphic, was also high in Prunus (63.9%). Almond and Japanese plum were the most variable among the diploid species (all but the hexaploid European plum) and peach the least polymorphic. Thirty-one microsatellites amplified and were polymorphic in all Prunus species studied, 12 of which, covering its whole genome, are proposed as the “universal Prunus set”. In contrast, only 16.3% were transferable in species of other Rosaceae genera (apple, pear, and strawberry). Polymorphic Prunus microsatellites also detected lower levels of variability in the non-congeneric species. No significant differences were detected in transferability and the ability to detect variability between microsatellites of EST and genomic origin.     

Microsatellite markers for diverse Salix species

Forty‐six microsatellites were isolated from an enriched library of Salix burjatica and tested on 20 individuals (of nine species/hybrids) from the National Willows Collection (IACR‐Long Ashton Research Station, UK). Twenty‐nine were monomorphic, gave multilocus or unscorable patterns, or were duplicates. The remaining 17 microsatellites gave 2–22 alleles/locus. Three microsatellites successfully cross‐amplified in 31 additional Salix species. A further six were tested on panels comprising 6–25 individuals from the 31 species. Cross‐amplification was successful in all cases. These results suggest that the microsatellites isolated here should prove useful for population studies in a wide range of Salix species.     

Isolation and Phylogenetic Analysis of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> from Its Invasive Alternative Host,Porcelain Berry

A strain of Xylella fastidiosa was isolated from an invasive alternative host species, porcelain berry. Its genetic relationship with strains isolated from a native alternative host, wild grape; a nonnative alternative host, mulberry; and other economically important hosts including cultivated grape, peach, plum, oak, maple and oleander was determined by using sequence analysis of the 16S–23S rRNA intergenic spacer region. Our phylogenetic analysis revealed that the porcelain berry strain is most closely related to the wild grape strain. These two strains are more closely related to the oak, peach, and plum strains than to the mulberry and oleander strains. They are separated from the maple and cultivated grape strains. Our data suggest that suppression of porcelain berry, wild grape, and mulberry in the vicinity of susceptible economically important hosts such as oak, peach, and plum may provide an important control measure for diseases caused by X. fastidiosa.     

Trans-specific <Emphasis Type="Italic">S</Emphasis>-RNase and SFB alleles in <Emphasis Type="Italic">Prunus</Emphasis> self-incompatibility haplotypes

Self-incompatibility in the genus Prunus is controlled by two genes at the S-locus, S-RNase and SFB. Both genes exhibit the high polymorphism and high sequence diversity characteristic of plant self-incompatibility systems. Deduced polypeptide sequences of three myrobalan and three domestic plum S-RNases showed over 97% identity with S-RNases from other Prunus species, including almond, sweet cherry, Japanese apricot and Japanese plum. The second intron, which is generally highly polymorphic between alleles was also remarkably well conserved within these S-allele pairs. Degenerate consensus primers were developed and used to amplify and sequence the co-adapted polymorphic SFB alleles. Sequence comparisons also indicated high degrees of polypeptide sequence identity between three myrobalan and the three domestic plum SFB alleles and the corresponding Prunus SFB alleles. We discuss these trans-specific allele identities in terms of S-allele function, evolution of new allele specificities and Prunus taxonomy and speciation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Seventy‐five EST‐linked Atlantic salmon (Salmo salar L.) microsatellite markers and their cross‐amplification in five salmonid species

An Atlantic salmon (Salmo salar L.) expressed sequence tag (EST) database consisting of 58 146 ESTs was screened for microsatellite sequences. Subsequent development of 75 polymorphic EST‐associated microsatellite markers in this species is described together with cross‐species amplification results of 133 gene‐associated tandem repeat markers in five salmonid species (Salmo trutta, Oncorhynchus mykiss, Salvelinus aplinus, Thymallus thymallus, Coregonus lavaretus). The number of alleles among EST‐linked microsatellites in Atlantic salmon ranged from two to 41 with an average of 12 alleles per locus. Cross‐species amplification resulted in detection of a total of 111 polymorphic locus‐species combinations (12–32 loci per species).     

Characterization of the major allergen of plum as a lipid transfer protein

Background: Allergy to Prunoideae fruit (plum, peach, cherry and apricot) is one of the most frequent food allergies in southern Europe. All these fruits cross-react in vivo and in vitro, as they share their major allergen, a 9 kD lipid transfer protein (LTP). Objective: The aim of the study was the identification and molecular characterization of the major allergen of plum. Methods: The IgE pattern of reactivity to plums was investigated by SDS–PAGE and immunoblotting with the sera of 23 patients. The identified major allergen was purified by HPLC, using a cationic-exchange column followed by gel-filtration. Further characterization was achieved by periodic-Schiff stain, isoelectrofocusing and N-terminal amino acid sequencing. Results and conclusions: The major allergen of plum is a 9 kD lipid transfer protein, not glycosylated and with a basic character (pI>9), highly homologous to the major allergen of peach.     

Identification and characterization of EST-SSRs and cpSSRs in endangered <Emphasis Type="Italic">Cycas hainanensis</Emphasis>

We report on the identification and characterization of six EST-linked simple sequence repeats (EST-SSRs) and chloroplast SSRs (cpSSRs) in endangered Cycas hainanensis. The number of alleles ranged from two to eight for EST-SSRs, two to three for cpSSRs. Observed and expected heterozygosities ranged from 0.042 to 0.417 and 0.042 to 0.811 for EST-SSRs, respectively. Expected heterozygosities ranged from 0.156 to 0.457 for cpSSRs. All these markers gave successful cross-species amplification in C. fairylakea. These markers will allow analyses of the baseline genetic variability and population structure of C. hainanensis to provide strategies for effective conservation and management. The experiments were carried out in South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, P.R. China.     

Transmission of prunus necrotic ringspot virus using plum pollen and thrips

Prunus necrotic ringspot virus (PNRSV) was transmitted to cucumber but not to peach seedlings after they were dusted with infective plum pollen and caged with 8–10 thrips per seedling for 24 h. When the pollen was taken from three plum trees shown by mechanical inoculation tests to have highly infective flower buds and pollen, the transmission rates to cucumber seedlings were 56% with Thrips tabaci and 66% with a mixture of five thrips species collected from Ageratum houstonianum flowers. However, the transmission rate averaged only 7% when pollen was taken from five other plum trees which had flowers with less infectivity in sap transmission tests. In 1990 T. imaginis, T. tabaci and T. australis, which were present in the mixture of thrips from A. houstonianum, also formed the major part of the thrips population in flowers of the plum trees used as the pollen source.     

Development of novel microsatellite markers for the grassland species Lolium multiflorum,Lolium perenne and Festuca pratensis

Twelve microsatellite markers were isolated from Lolium multiflorum. Allelic variability and cross‐species amplification were assessed on 16 individuals of each of the three grassland species L. multiflorum, Lolium perenne and Festuca pratensis. Cross‐species amplification success was 100% for L. perenne and 83% for F. pratensis. The number of alleles detected ranged from one to 14 with an average of 3.4. While three microsatellite loci were polymorphic in all three species, one marker produced species‐specific alleles in all three species. These microsatellite markers provide a valuable tool for population genetic studies within and among species of the Festuca–Lolium complex.     

Isolation of microsatellite loci from spotted gum (Corymbia variegata), and cross‐species amplification in Corymbia and Eucalyptus

Corymbia variegata (spotted gum) is an important commercial hardwood timber species in Australia. Fourteen polymorphic microsatellite loci were isolated from C. variegata, with 3–5 alleles amplified in three individuals examined. Cross‐species amplification in Corymbia was successful for all primer pairs, while 10 loci (71%) were successfully transferred to at least one species in the closely related genus Eucalyptus.     

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.     

Host preference of the plum curculio

We assessed host preference of adult plum curculio, Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae), based on the total number of mark‐released and wild adults recovered and the total distance moved by mark‐released adults in an orchard whose layout was designed to specifically allow foraging plum curculios to choose among host tree species. Host trees included apple, Malus domestica Borkh.; pear, Pyrus communis (L.); peach, Prunus persica (L.) Batsch; apricot, Prunus armeniaca L.; tart cherry, Prunus cerasus L.; sweet cherry, Prunus avium (L.); European plum, Prunus domestica L.; and Japanese plum, Prunus salicina Lindl. (all Rosaceae). We released 2900 marked adults and recovered 17.7%. We used screen traps to provide a measure of the number of adults that arrived at and climbed up particular host trees and found that significantly greater numbers of marked adults and the greatest number of wild adults were recovered from screen traps attached to Japanese plum. We sampled host tree canopies by tapping limbs to provide a measure of the number of adults within a tree canopy at a particular moment. Again, significantly greater numbers of marked and wild adults were recovered from plum species, with no difference between Japanese and European plum cultivars for marked individuals, but with significantly greater numbers of wild individuals recovered from Japanese plum. The preference index (PI) for Japanese plum based on total distances moved by all marked adults recovered on Japanese plum divided by the total distance moved by marked adults recovered on other host trees indicated that Japanese plum was the most highly preferred host, followed by European plum, peach, sweet cherry, tart cherry, apricot, apple, and pear, respectively.     

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.