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, 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.     

Simple‐sequence repeat (SSR) markers of Japanese plum (Prunus salicina Lindl.) are highly polymorphic and transferable to peach and almond

Thirty‐five polymorphic microsatellites were developed using a CT/AG enriched genomic library of Japanese plum cv. Santa Rosa. Twenty‐seven of them detected a single locus and eight two or more loci. A high level of variability was observed in a set of eight cultivars for the 27 single‐locus microsatellites: 5.7 average number of alleles per locus; 73% mean observed heterozygosity and 74% discrimination power. Most SSRs were transferable to peach (85%) and almond (78%).     

Detection of genetic diversity in closely related bread wheat using microsatellite markers

Wheat microsatellites (WMS) were used to estimate the extent of genetic diversity among 40 wheat cultivars and lines, including mainly European elite material. The 23 WMS used were located on 15 different chromosomes, and revealed a total of 142 alleles. The number of alleles ranged from 3 to 16, with an average of 6.2 alleles per WMS. The average dinucleotide repeat number ranged from 13 to 41. The correlation coefficient between the number of alleles and the average number of repeats was only slight (r _s = 0.55). Based on percentage difference a dendrogram is presented, calculated by the WMS-derived data. All but two of the wheat cultivars and lines could be distinguished. Some of the resulting groups are strongly related to the pedigrees of the appropriate cultivars. Values for co-ancestry (f) of 179 pairs of cultivars related by their pedigrees (f0.1) averaged 0.29. Genetic similarity (GS) based on WMS of the same pairs averaged 0.44. The rank correlation for these pairs was slight, with r _s = 0.55, but highly significant (P<0.001). The results suggest that a relatively small number of microsatellites can be used for the estimation of genetic diversity and cultivar identification in elite material of hexaploid bread wheat.     

Microsatellite genotyping of carnation varieties

A set of 11 sequence-tagged microsatellite markers for carnation (Dianthus caryophyllus) was developed using a DNA library enriched for microsatellites. Supplemented with three markers derived from sequence database entries, these were used to genotype carnation varieties using a semi-automated fluorescence-based approach. In a set of 82 cultivars, the markers amplified 4-16 alleles each. The effective number of alleles varied from 1.9 to 6.0. For the eight best scorable markers, heterozygosity was between 0.51 and 0.99. The markers were able to distinguish all cultivars with a unique combination of alleles, except for sport mutants, which were readily grouped together with the original cultivar. In addition, one group of three and one group of six cultivars each had the same combination of 'allelic peaks'. The cluster of three varieties concerned original cultivars and their mutants. The cluster of six consisted of four mutants from the same cultivar and two other varieties.     

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.     

Microsatellites from kousa dogwood (Cornus kousa)

Microsatellite loci were identified from Cornus kousa'National'. Primer pairs for 86 loci were developed and of these, eight were optimized and screened using genomic DNA from 22 kousa cultivars. All optimized loci were polymorphic and the number of alleles per locus ranged from three to 17. Observed heterozygosity ranged from 0 to 0.3 and expected heterozygosity ranged from 0.38 to 0.91. These microsatellites will be useful in population studies, and a breeding programme for cultivar development of Cornus species.     

Characterization of Indian bred rose cultivars using morphological and molecular markers for conservation and sustainable management

Rose (Rosa × hybrid L.) is one of the most important commercial ornamental crops cultivated worldwide for its beauty, fragrance and nutraceutical values. Characterization of rose germplasm provides precise information about the extent of diversity present among the cultivars. It also helps in cultivar identification, intellectual property right protection, variety improvement and genetic diversity conservation. In the present study, 109 Indian bred rose cultivars were characterized using 59 morphological and 48 SSR markers. Out of 48 SSRs used, 31 markers exhibited polymorphism and 96 alleles were identified with an average of 3.9 alleles per locus. Nei’s expected heterozygosity value of each locus ranged from 0.08 (with SSR ABRII/RPU32) to 0.78 (SSR Rh58). The similarity coefficient values ranged from 0.42 to 0.90 which indicated presence of moderated diversity among Indian cultivars. The neighbor-joining tree based on morphological data grouped the cultivars into two major clusters and several minor clusters based on their morphological resemblance. However, UPGMA dendrogram constructed using matching coefficient values grouped the cultivars into eight different clusters. Interpopulation analysis revealed higher genetic similarities between Hybrid Tea and Floribunda cultivars. An analysis for presence of population sub-structure grouped the Indian cultivars into eight different genetic groups. Analysis of molecular variance revealed apportioning of 97.59% of the variation to within subgroup diversity and 3.07% to between the cultivar groups. We have demonstrated here successful utilization of robust SSR to distinguish cultivars and assess genetic diversity among Indian bred rose cultivars. The information provided here is useful for cultivar identification and protection, cultivar improvement and genetic diversity conservation.     

Nuclear- and chloroplast-microsatellite variation in A-genome species of rice.

Simple sequence length polymorphism analysis was carried out to reveal microsatellite variation and to clarify the phylogenetic relationships among A-genome species of rice. Total DNA from 29 cultivars (23 Oryza sativa and 6 O. glaberrima) and 30 accessions of wild A-genome species (12 O. rufipogon, 5 O. glumaepatula, 2 O. longistaminata, 6 O. meridionalis, and 5 O. barthii) was used as a template for PCR to detect 24 nuclear and 10 chloroplast microsatellite loci. Microsatellite allelic diversity was examined based on amplified banding patterns. Microsatellites amplified clearly in all 59 accessions, with an average of 18.4 alleles per locus. The polymorphism information content (PIC) value ranged from 0.85 to 0.94, with an average of 0.89. At the species level, high average PIC values were observed in O. sativa (0.79) and O. rufipogon (0.80). For chloroplast microsatellites, the average number of alleles per locus and the average PIC value were 2.9 and 0.38, respectively. While the magnitude of diversity was much greater for nuclear microsatellites than for chloroplast microsatellites, they showed parallel patterns of differentiation for each taxonomic group. Using the ratio of common alleles (estimated as size of amplified fragments) as a similarity index, the average percentages of common microsatellite alleles were calculated between taxa. For both nuclear and chloroplast microsatellites, O. sativa showed the highest similarity values to O. rufipogon, and O. glaberrima was most similar to O. barthii. These data support previous evidence that these cultivars originated from the corresponding wild ancestral species.     

Development of SSR markers for the phylogenetic analysis of almond trees from China and the Mediterranean region.

Expressed sequence tag (EST) derived simple sequence repeats (SSRs, microsatellites) were screened and identified from 3863 almond and 10 185 peach EST sequences, and the spectra of SSRs in the non-redundant EST sequences were investigated after sequence assembly. One hundred seventy-eight (12.07%) almond SSRs and 497 (9.97%) peach SSRs were detected. The EST-SSR occurs every 4.97 kb in almond ESTs and 6.57 kb in peach, and SSRs with di- and trinucleotide repeat motifs are the most abundant in both almond and peach ESTs. Twenty one EST-SSRs were thereafter, developed and used together with 7 genomic SSRs, to study the genetic relationship among 36 almond (P. communis Fritsch.) cultivars from China and the Mediterranean area, as well as 8 accessions of other related species from the genus Prunus. Both EST-derived and genomic SSR markers showed high cross-species transferability in the genus. Out of the 112 polymorphic alleles detected in the 36 cultivated almonds, 28 are specific to Chinese cultivars and 25 to the others. The 44 accessions were clustered into 4 groups in the phylogenetic tree and the 36 almond cultivars formed two distinct subgroups, one containing only Chinese cultivars and one of unknown origin and the other only those originating from the Mediterranean area, indicating that Chinese almond cultivars have a distinct evolutionary history from the Mediterranean almond. Our preliminary results indicated that common almond was more closely related to peach (P. persica (L.) Batsch.) than to the four wild species of almond, (P. mongolica Maxim., P. ledebouriana Schleche, P. tangutica Batal., and P. triloba Lindl.). The implications of these SSR markers for evolutionary analysis and molecular mapping of Prunus species are discussed.     

Development and characterization of microsatellite markers in Indian sesame (Sesamum indicum L.)

The genetic characterization of Indian sesame cultivars and related wild species was analysed using 102 simple sequence repeat (SSR; microsatellite) markers. Of these, 62 were novel sesame-specific microsatellites isolated in the course of the present investigation by constructing genomic libraries. Characterization of the 68 sesame accessions and three related wild species using 72 polymorphic SSR primers resulted in the detection of 170 alleles. The number of alleles ranged from two to four with an average of 2.5 alleles per locus. Polymorphic information content of the markers ranged from 0.43 to 0.88 with an average of 0.66. UPGMA cluster analysis grouped all the accessions into two major clusters with a genetic similarity ranging from 0.40 to 0.91. A moderate to high level of genetic variability was observed. The three wild accessions used in the study formed separate clades and distant genetic relationships were observed between the cultivar lines and wild species. Differentiation of genotypes according to geographical region was not observed. Analysis of molecular variance (AMOVA) analysis revealed that a high percentage of variation was within populations (87.1 %). An overall F _st of 0.11 among the populations indicated low population differentiation. The SSR markers developed will be useful for further genetic analysis, linkage mapping and selection of parents in future breeding programmes.     

Development and characterization of microsatellites of an important medicinal plant Orthosiphon stamineus (misai kucing)

Orthosiphon stamineus (Lamiaceae) is an important medicinal plant in Malaysia that has great potential in the herbal medicine industry. In this study, we developed and characterized ten microsatellites for O. stamineus. The microsatellite loci were checked for variability using 28 individuals of O. stamineus sampled from 10 nurseries. The number of alleles ranged from two to four, and the observed heterozygosity ranged from 0.143 to 0.963. Neighbor-joining analysis revealed two main genetic clusters corresponding to the two varieties (white and purple). The application of these microsatellites will facilitate the cultivar/variety identification and germplasm management in the species.     

Genetic variability of introduced and local Spanish peach cultivars determined by SSR markers

A set of 94 peach cultivars including Spanish native peach and foreign commercial cultivars were analyzed using 15 SSR markers, selected for their high level of polymorphism. The number of alleles obtained varied from two to 11 with an average of 6.73 giving 185 different genotypes. All the cultivars showed a unique genetic profile, each one using different genotypic combination of all loci. BPPCT001 was the most informative locus showing also the highest discrimination power. Only six loci allowed the unambiguous separation of all the Spanish native cultivars studied, and the genotypic combination of only eight loci permitted the total differentiation of the 94 peach cultivars analyzed. The six selected loci (BPPCT001, BPPCT006, BPPCT008, PS9f8, UDP98-022, and UDP98-412) seem to be very useful for future Spanish peach identification works, and they will help to establish a molecular data base for native peach cultivars. UPGMA analysis was performed from the genetic distance matrix, and allowed the arrangement of all genotypes according to their genetic diversity. The genetic diversity among cultivars, observed in this work, led to their separation according to their regional origin, their morphological characteristics, and especially according to their fruit traits. Analysis of molecular variance was performed for seven populations from different regions of Spain and USA to examine the distribution of genetic variation of the studied accessions, showing that the major variation occurred within populations in each geographic site. The results reveal the existence of two diversity regions in Spain for peach germplasm.     

Seasonal patterns of reproductive and vegetative sink activity in early and late maturing peach (Prunus persica) cultivars

Weekly measurements of fruit growth, fruit respiration and shoot extension growth were made in the field on early (June Lady) and late (O'Henry) maturing cultivars of peach ( Prunus persica L. Batsch). The seasonal patterns of fruit growth and respiration for the two cultivars were very similar except that the early maturing cultivar bloomed a few days earlier than the late cultivar and had a shorter intermediate stage (Stage II) of fruit growth. Maximum rates of fruit respiration per unit weight at 20°C were similar for both cultivars during the first two stages of fruit growth but higher for the early cultivar during the final stage of fruit growth. Maximum fruit growth rates within any particular stage of fruit growth were similar for both cultivars, but the mean fruit weight of the late cultivar was greater at the end of Stage II, because of the extended length of this stage compared to the early cultivar. The final stage of most rapid fruit growth and respiration coincided with the period of most rapid shoot extension growth in the early maturing cultivar but occurred after this period in the late maturing cultivar. Genetic selection for early fruit maturity in peach has apparently had little effect on timing of shoot growth and this may result in increased competition between vegetative and reproductive sinks during peak periods of fruit growth in early maturing cultivars.     

Mapping osmotic adjustment in an advanced back-cross inbred population of rice

Osmotic adjustment is one of several characters putatively associated with drought tolerance in rice. Indica cultivars are known to have a greater capacity for osmotic adjustment than japonica cultivars. We developed an advanced back-cross population using an indica donor, IR62266-42-6-2, to introgress osmotic adjustment into an elite japonica cultivar, IR60080-46A. One hundred and fifty BC₃F₃ families were genotyped using microsatellites and RFLP markers, and a few candidate genes. We evaluated osmotic adjustment in these lines under greenhouse conditions using the re-hydration technique. Using the composite interval mapping technique, we detected 14 QTLs located on chromosomes 1, 2, 3, 4, 5, 7, 8 and 10 that together explained 58% of the phenotypic variability. Most, but not all, of the alleles with positive effects came from the donor parent. On chromosome 8, two QTLs were associated in repulsion. The QTL locations were in good agreement with previous studies on this trait on rice and in other cereals. Some BC₃F₃ lines carried the favorable alleles at the two markers flanking up to four QTLs. Intercrossing these lines followed by marker-aided selection in their progenies will be necessary to recover lines with levels of osmotic adjustment equal to the donor parent. The advanced back-cross strategy appeared to be an appropriate method to accelerate the process of introgressing interesting traits into elite material.Communicated by Q. Zhang     

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.     

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.     

The use of microsatellites to analyze relationships and to decipher homonyms and synonyms in Azorean apples (Malus?×?domestica Borkh.)

The objective of this study was to exploit the molecular and morphological variability present in Malus domestica to clarify the confused characterization of apple plantations in the Azores. Most Azorean apples are grown in orchards. They are usually given a local name, and sometimes the same name is used for different cultivars and varieties which share morphology and should be known by different names. Two-hundred samples of apples cultivated in the Azores were analyzed by use of ten microsatellites. The total number of alleles per locus ranged from 10 to 24, with a mean of 15.2. Heterozygosity was high, reflecting the high variability of the samples. Expected heterozygosity varied within a narrow range, from 0.74 to 0.88, whereas observed heterozygosity ranged from 0.41 to 0.98. The high genetic variability contributed to the high discriminating power, which ranged between 0.84 and 0.93. These microsatellites were used to unambiguously discriminate most of the tested apple cultivars on the basis of their allelic profiles. The rooted UPGMA tree organized most of the samples into thirteen main clusters, often with high bootstrap values. We identified 60 synonyms and 32 homonyms among the samples. Moreover, it was possible to relate each individual to its originating population and detect likely parent–offspring groups.     

Fluorescence-based AFLPs occur as the most suitable marker system for oilseed rape cultivar identification

Three different types of molecular markers, RAPD, SSR and fluorescence-based AFLP, were evaluated and compared for their ability to identify oilseed rape cultivars. The direct comparison of RAPD, SSR and AFLP approaches in cultivar identification showed that the AFLP methodology detected polymorphisms more efficiently than either RAPD or SSR methods. For the characterisation of six oilseed rape cultivars, 60 RAPD primers were tested and only eight of them (14%) detected sufficient levels of polymorphism. Five microsatellites out of fifteen tested were polymorphic, but in all loci, except one, only two different alleles were detected. This result indicated the limited degree of polymorphism found in Brassica napus. Each of the six tested AFLP combinations detected polymorphisms, the best combination (M-CAA/E-ACT) had 26% polymorphic peaks from a total of 90 peaks and could distinguish the analysed cultivars and 4 out of 5 core lines of cultivars. The results presented show that florescence-based AFLP is, for the purposes of oilseed rape cultivar fingerprinting, a more suitable approach than either RAPD or SSR.     

Molecular characterisation of sweet cherry (Prunus avium L.) genotypes using peach [Prunus persica (L.) Batsch] SSR sequences

A total of 76 sweet cherry genotypes were screened with 34 microsatellite primer pairs previously developed in peach. Amplification of SSR loci was obtained for 24 of the microsatellite primer pairs, and 14 of them produced polymorphic amplification patterns. On the basis of polymorphism and quality of amplification, a set of nine primer pairs and the resulting 27 informative alleles were used to identify 72 genotype profiles. Of these, 68 correspond to unique cultivar genotypes, and the remaining four correspond to three cultivars that could not be differentiated from the two original genotypes of which they are mutants, and two very closely related cultivars. The mean number of alleles per locus was 3.7 while the mean heterozygosity over the nine polymorphic loci averaged 0.49. The results demonstrate the usefulness of cross-species transferability of microsatellite sequences allowing the discrimination of different genotypes of a fruit tree species with sequences developed in other species of the same genus. UPGMA cluster analysis of the similarity data divided the ancient genotypes studied into two fairly well-defined groups that reflect their geographic origin, one with genotypes originating in southern Europe and the other with the genotypes from northern Europe and North America.