Isolation and characterization of polymorphic microsatellites in diploid strawberry (Fragaria vesca L.) for mapping,diversity studies and clone identification

This study reports the development and characterization of 10 polymorphic microsatellite primer pairs in wild strawberry (Fragaria vesca). The primers were designed from a genomic library enriched for di‐, tri‐ and tetranucleotide repeats from F. vesca‘Reugen’. They showed single locus polymorphism in a set of nine F. vesca accessions; two to six alleles were detected per locus. The level of polymorphism in F. vesca was surprisingly low, although three pairs of primers were sufficient to distinguish between most accessions.     

Microsatellite (SSR) markers reveal genetic identities, genetic diversity and relationships in a Malus×domestica borkh. core subset collection

 A collection of 66 Malus×domestica Borkh. accessions from the USDA-ARS Plant Genetic Resources Unit’s core collection was screened with a set of eight SSR (simple sequence repeat) primers developed at the PGRU in order to determine genetic identities, estimate genetic diversity, and to identify genetic relationships among these accessions. All eight primer pairs generated multiple fragments when used in amplification reactions with DNA from these accessions. High levels of variation were detected with a mean of 12.1 alleles per locus and a mean heterozygosity across all eight loci of 0.693. The eight primer pairs utilized in this study unambiguously differentiated all but seven pairs of accessions in this collection of 66 M.×domestica Borkh. genotypes. The probability of matching any two genotypes at all eight loci in this study was approximately 1 in 1 billion. The markers detected two misnamed accessions in the collection. Genetic-identity data produced a genetic-relatedness phenogram which was concordant with geographic origins and/or known pedigree information. These SSR markers show great promise as tools for managing Malus ex situ germplasm collections as well as for collection and preservation strategies concerning wild Malus populations in situ. Received: 28 March 1998 / Accepted: 29 April 1998     

The development of eight microsatellite loci in the wild daffodil Narcissus triandrus (Amaryllidaceae)

We report microsatellite primer pairs for the wild tristylous daffodil, Narcissus triandrus (Amaryllidaceae). From enriched libraries, we identified 58 unique microsatellite loci. We designed primer pairs for 27 of these loci and screened genomic DNA from 38 to 40 adults from a single population. For eight polymorphic loci, the number of alleles per locus ranged from five to 17. As six primers also amplified loci in three other Narcissus species, including two horticultural varieties, we expect that some of these markers will be transferable to other Narcissus species.     

Development of microsatellite primers and two multiplex polymerase chain reactions for the common elder (Sambucus nigra)

Primer pairs were developed for eight microsatellite loci isolated from a genomic DNA library of Sambucus nigra‘Black Beauty’. In a survey of 21 accessions of S. nigra, all revealed polymorphism and the number of alleles per locus ranged from three to 18. Two multiplex polymerase chain reactions (PCRs) were optimized and these discriminated all the accessions tested providing a cost‐effective genotyping solution for the species. Six primer pairs (EMSn002, 003, 010, 016, 017, and 023) were informative in two Sambucus canadensis accessions.     

Genetic Diversity of Some Pear Cultivars and Genotypes Using Simple Sequence Repeat (SSR) Markers

The genetic diversity and relationships among 47 pear cultivars and genotypes (Pyrus spp.), including 4 Japanese pears (Pyrus pyrifolia), 40 European pears (Pyrus communis), 1 Chinese pear (Pyrus bretschneideri) as well as 2 wild relatives (Pyrus salicifolia and Pyrus mazandaranica) were studied using 28 microsatellite primer pairs. A total of 174 alleles were produced at the 28 SSR loci with their sizes ranging from 81 to 290?bp. The number of observed alleles for each locus ranged from 3 (TsuENH014 and TsuENH046) to 12 (NB103a), with an average of 6.21 alleles per locus. In some SSR loci, more than two alleles were amplified in some cultivars and genotypes, suggesting that duplication has occurred in those accessions. This information suggests that at least two genomic regions exist for these loci in the pear genome. The observed heterozygosity (H _o) values of amplified loci ranged from 0.17 (TsuENH006) to 0.97 (NB103a). Shannon's information index (I) value was observed to be highest (2.14) in the NB103a locus, while the TsuENH006 locus had the lowest value with an average of 1.37 among SSR loci. The Dice genetic similarity coefficient ranged from 0.29 (??Nijisseiki?? and P. mazandaranica) to 0.91 (??Chojuro?? and ??Nijisseiki??) among samples. UPGMA cluster analysis showed two major groups corresponding to the Japanese and European pears.     

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 of “universal” gene-specific markers from Malus spp. cDNA sequences, their mapping and use in synteny studies within Rosaceae

The Rosaceae contains many economically valuable crop genera, including Malus (apple), Fragaria (strawberry), and Prunus (stone fruit). There has been increasing interest in the development of linkage maps for these species, with a view to marker-assisted selection to assist breeding programs and, recently, in the development of transferable markers to permit syntenic comparisons of maps of different rosaceous genera. In this investigation, a set of Malus cDNA sequences were downloaded from the European Molecular Biology Laboratory database. The sequences were aligned with homologous full-length Arabidopsis genomic DNA sequences to identify putative intron–exon junctions and conserved flanking exon sequences. Primer pairs were designed from the conserved exon sequences flanking predicted intron–exon junctions in the Malus cDNA sequences. These were used to amplify products by polymerase chain reaction from the parents of the Malus mapping progeny “Fiesta” × “Totem.” Eleven loci, representing ten genes (39%), were polymorphic in the “Fiesta” × “Totem” population and mapped to seven Malus linkage groups. Transferability to other rosaceous genera was high, with primer pairs representing 85% of genes, amplifying products from Fragaria and primer pairs representing 85% of genes, amplifying products from Prunus genomic DNA. These primers were screened in the Fragaria and Prunus mapping bin sets and 38% of the genes were successfully located on both maps. Analysis of the markers mapped in more than one rosaceous genus revealed patterns of synteny between genera, while a comparison with the physical positions of homologous genes on the Arabidopsis genome revealed high sequence conservation but only fragmentary patterns of macrosynteny.     

The development and mapping of functional markers in Fragaria and their transferability and potential for mapping in other genera

We have developed 46 primer pairs from exon sequences flanking polymorphic introns of 23 Fragaria gene sequences and one Malus sequence deposited in the EMBL database. Sequencing of a set of the PCR products amplified with the novel primer pairs in diploid Fragaria showed the products to be homologous to the sequences from which the primers were originally designed. By scoring the segregation of the 24 genes in two diploid Fragaria progenies FV × FN (F. vesca × F. nubicola F₂) and 815 × 903BC (F. vesca × F. viridis BC₁) 29 genetic loci at discrete positions on the seven linkage groups previously characterised could be mapped, bringing to 35 the total number of known function genes mapped in Fragaria. Twenty primer pairs, representing 14 genes, amplified a product of the expected size in both Malus and Prunus. To demonstrate the applicability of these gene-specific loci to comparative mapping in Rosaceae, five markers that displayed clear polymorphism between the parents of a Malus and a Prunus mapping population were selected. The markers were then scored and mapped in at least one of the two additional progenies.     

Isolation and characterization of microsatellite loci from the orchid Serapias vomeracea (Orchidaceae) and cross‐priming to other Serapias species

In this paper we describe the isolation and characterization of six polymorphic microsatellite loci from the orchid Serapias vomeracea. This species is widely distributed in the Mediterranean region. Microsatellite loci were isolated from an enriched library and primer pairs were designed for 18 loci. Primer pairs for six loci amplified well and were tested on samples from southern Italy. Levels of genetic variability detected at these six loci are high, with numbers of alleles per locus ranging from 3 to 6, and observed heterozygosity (H_O) ranging from 0.35 to 0.86. All primer pairs tested amplified DNA from four other Serapias species, indicating that the primers are useful for population genetic studies throughout the genus.     

Identification and characterization of microsatellite loci in the common fig (Ficus carica L.) and representative species of the genus Ficus

We developed microsatellites in fig (Ficus carica L.). A TC and TG‐enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Eight primer pairs produced amplification products that were both interpretable and polymorphic in 14 fig cultivars and two French wild‐growing populations of F. carica (n₁ = 9 and n₂ = 10). Number of alleles per locus ranged from three to six. Except for one microsatellite locus, the observed heterozygosity was higher than the expected value. The F. carica microsatellites gave amplification products in 17 other Ficus species in 86% of the cases.     

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.     

SSRs isolated from apple can identify polymorphism and genetic diversity in pear

Apple simple sequence repeats (SSRs) were intergenerically applied to the characterization of 36 pear accessions, including 19 Japanese pears (Pyrus pyrifolia), 7 Chinese pears (P. bretschneideri, P. ussuriensis), 5 European pears (P. communis), 3 wild relatives (P. calleryana), and 2 hybrids between P. pyrifolia and P. communis. All of the tested SSR primers derived from apple produced discrete amplified fragments in all pear accessions. Nucleotide repeats were detected in the amplified bands by both Southern blot and sequencing analysis, and nucleotide sequences of pear were compared with those of apple. The differences in fragment size among pear or between pear and apple were, in many cases, due to the differences in repeat number. Interestingly, the DNA sequence of flanking regions in apple was highly conserved in pear. Hybrids from P. pyrifolia×P. communis showed one fragment inherited from each parent in all scorable cases, which suggested that each primer pair amplified fragments originating from the same locus. A total of 79 alleles were detected from seven SSR loci in pear, and all pear varieties except for the mutants could be differentiated. In conclusion, SSRs isolated from apple are highly conserved in pear and could be utilized as DNA markers in the latter genus. Received: 17 July 2000 / Accepted: 22 September 2000     

Isolation and characterization of novel polymorphic nuclear microsatellite markers from Ophrys fusca (Orchidaceae) and cross-species amplification

The present study reports the isolation and characterization of eight new polymorphic microsatellite loci from the sexually deceptive orchid Ophrys fusca. Microsatellites were isolated from two partially enriched genomic libraries using FIASCO (Fast Isolation by AFLP of Sequences COntaining repeats). Seventy-three loci were screened for primer design and primer pairs corresponding to eight different loci were selected for microsatellite characterization of two Portuguese populations. Total number of alleles per locus ranged from 10 to 32. All loci showed a high level of observed heterozygosity (H₀) ranging from 0.33 to 1 and were possible to amplify in 16 other species of Ophrys using the same primers. H. C. Cotrim and F. A. Monteiro have contributed equally to this work.     

Isolation and characterization of microsatellite markers in the sacred lotus (Nelumbo nucifera Gaertn.)

The sacred lotus (Nelumbo nucifera Gaertn.) is an aquatic plant of economic and ornamental importance in China. From an (AG)_n‐enriched genomic library, 24 microsatellites were isolated and identified by using the (fast isolation by the AFLP of sequences containing repeats) FIASCO protocol. Eleven loci showed polymorphism with two to six alleles per locus. These markers yielded 42 alleles in a survey of 32 accessions of the sacred lotus. Eleven effective primer pairs of simple sequence repeats were designed and will be used as genetic markers to evaluate the fine‐scale population structure of the sacred lotus in the future.     

Development and genetic mapping of 127 new microsatellite markers in barley

To enhance the marker density of existing genetic maps of barley (Hordeum vulgare L.), a new set of microsatellite markers containing dinucleotide motifs was developed from genomic clones. Out of 254 primer pairs tested, a total of 167 primer pairs were classifed as functional in a panel of six barley cultivars and three H. spontaneum accessions, and of those, 127 primer pairs resulting in 133 loci were either mapped or located onto chromosomes. The polymorphism information content (PIC) ranged from 0.05 to 0.94 with an average of 0.60. The number of alleles per locus varied from 1 to 9. On average, 3.9 alleles per primer pair were observed. The RFLP frameworks of two previously published linkage maps were used to locate a total of 115 new microsatellite loci on at least one mapping population. The chromosomal assignment of 48 mapped loci was corroborated on a set of wheat-barley chromosome addition lines; 18 additional loci which were not polymorphic in the mapping populations were assigned to chromosomes by this method. The microsatellites were located on all seven linkage groups with four significant clusters in the centromeric regions of 2H, 3H, 6H and 7H. These newly developed microsatellites improve the density of existing barley microsatellite maps and can be used in genetic studies and breeding research.Communicated by G. Wenzel     

Characteristics of single- and multi-copy microsatellites from Pinus radiata

 Dinucleotide microsatellites were isolated from Pinus radiata using both a standard genomic library and libraries enriched for microsatellites. Locus-specific primers were designed to amplify 43 unique microsatellites. Thirty two of these loci had interpretable PCR patterns, 11 of which were polymorphic in a screen of 19 P. radiata individuals; all 11 polymorphic loci contained at least 17 repeats in the sequenced plasmid. Six of the eleven primer pairs amplified multiple fragments per individual (3–8), suggesting that these loci were present in multiple copies in the genome. Genotyping a 48-tree P. radiata production population with seven of the most polymorphic microsatellites revealed an average of 17 bands per locus (the multi-copy microsatellites were treated as one locus). When tested on known pedigrees, both single and multi-copy microsatellites exhibited co-dominant inheritance and Mendelian segregation. Two loci had null alleles and one locus had a high frequency of non-parental alleles, suggesting a high mutation rate. Eight of these microsatellites, including five multi-copy loci, were placed on a partially constructed P. radiata genetic map. Four of the five multi-copy microsatellites had two or more sets of alleles that mapped to the same locus, and the fifth mapped to two unlinked loci. All seven tested primer pairs amplified PCR products from other species of hard pine, three amplified products from soft-pine species, and one amplified bands in other conifers. Received: 10 November 1997 / Accepted: 5 January 1998     

Polymorphic microsatellite primers developed from DNA of the endangered Mekong giant catfish,Pangasianodon gigas (Chevey) and cross‐species amplification in three species of Pangasius

The critically endangered Pangasianodon gigas is endemic to the Mekong River. Despite its importance, little is known about its genetic diversity and conservation efforts are hampered. Ten polymorphic dinucleotide microsatellite primer pairs were developed from DNA of P. gigas. The analysis of 20 individuals from hatchery stocks using these primers resulted in two to six alleles/locus; H_O = 0.05–0.95; H_E = 0.05–0.81. All but one locus (Pg‐3) conformed to Hardy–Weinberg expectation. Eight, six and seven primer pairs were amplified with the DNA from Pangasianodon hypophthalmus, Pangasius larnaudii and Pangasius sanitwongsei, respectively. These markers will be useful for genetic monitoring of wild and hatchery stocks of these pangasiids.     

Development of a locus-specific,co-dominant SCAR marker for assisted-selection of the <Emphasis Type="Italic">Sw-5</Emphasis> (<Emphasis Type="Italic">Tospovirus</Emphasis> resistance) gene cluster in a wide range of tomato accessions

The best levels of broad-spectrum Tospovirus resistance reported in tomatoes thus far are conferred by the Sw-5 locus. This locus contains at least five paralogues (denoted Sw-5a through Sw-5e), of which Sw-5b represents the actual resistance gene. Here we evaluated a panel of seven PCR primer pairs matching different sequences within a genomic region spanning the Sw-5a and Sw-5b gene cluster. Primer efficiency evaluation was done employing tomato isolines with and without the Sw-5 locus. One primer pair produced a single and co-dominant polymorphism between susceptible and resistant isolines. Sequence analysis of these amplicons indicated that they were specific for the Sw-5 locus and their differences were due to insertions/deletions. The polymorphic SCAR amplicon encompass a conserved sequence of the promoter region of the functional Sw-5b gene, being located in the position −31 from its open reading frame. This primer pair was also evaluated in field assays and with a collection of accessions known to be either susceptible or resistant to tospoviruses. An almost complete correlation was found between resistance under greenhouse/field conditions and the presence of the marker. Therefore, this primer pair is a very useful tool in marker-assisted selection systems in a large range of tomato accessions.     

Microsatellite primers for Sorbus torminalis and related species

This study reports the cloning and characterization of nine microsatellite primer pairs in a scattered woody species (Sorbus torminalis), and shows their potential for further use in 36 species of the Maloideae, a Rosaceae subfamily containing important fruit crop and ornamental species. These primers were designed from a microsatellite library constructed from genomic DNA of S. torminalis and enriched for CA and GA repeats. Genotyping 48 S. torminalis of a natural population with the six best markers yielded a mean of 10.7 alleles per locus, and an expectation of exclusion probability for paternity analysis greater than 0.993.