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     

Increasing the Efficiency of Microsatellite Discovery from Poorly Enriched Libraries in Coniferous Forest Species

Microsatellites are PCR based markers widely applicable in many plant species. Although the discovery and development of new microsatellites is a costly and technically demanding process, they are widely used in many plant systems. In coniferous tree species, the large genome size and repetitive DNA regions further aggravate the process, often resulting in libraries with very low enrichment efficiencies. Such species have proven intractable to efficient microsatellite discovery. For example, enriched microsatellite libraries produced for seven angiosperm species had enrichment efficiencies up to 50%, whereas libraries for three conifers, generated concurrently, under identical conditions, all had enrichment efficiencies below 10%. This report describes a simple strategy for recovering useful microsatellites from two of these conifer libraries, Pinus elliottii and Araucaria cuninghamii. Enrichment efficiencies increased from 0% to 100% and 1.5% to 98%, respectively. The strategy utilised DIG labelled oligo probes to identify clones containing microsatellite inserts. The technique is cost effective and rapid, utilising basic techniques and equipment that are widely available.     

Development of a set of highly polymorphic genomic microsatellites (gSSRs) in Sitka spruce (Picea sitchensis (Bong.) Carr.)

Robust, polymorphic microsatellite DNA markers (simple sequence repeats—SSRs) are valuable tools for a range of tree conservation and breeding applications. SSRs are routinely used in the study of population genetic structure and diversity, pedigree reconstruction and genetic linkage mapping. Their abundance in the genome, co-dominant inheritance and potential for cross-species amplification make microsatellites highly prized markers. This paper characterises 22 novel genomic polymorphic microsatellite loci for Sitka spruce (Picea sitchensis (Bong.) Carr.). Amplification of DNA from Sitka spruce material was carried out both with a set of unrelated trees to obtain diversity statistics for each locus, and with the progeny of a full-sib family to test simple Mendelian inheritance. Observed heterozygosity ranged from 0.38 to 0.91 and allele number per locus ranged from 6 to 21, with a mean of 12.2. In addition, the primer pairs were tested with DNA from Norway spruce (P. abies) and white spruce (P. glauca) to investigate their potential for cross-species amplification and ten loci amplified in all three species. The results from these genomic microsatellites are compared to data generated from microsatellites derived from Picea EST libraries. In summary, this novel, highly polymorphic markers represent a significant addition to the rapidly expanding Picea genomics tool-box. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An improved method for the isolation of total RNA from spurce tissues

After many unsuccessful attempts to obtain biologically active mRNAs from spruce (Picea abies) tissues using available protocols, we have adapted a procedure for the isolation of RNAs from needles, shoots, and callus ofPicea species. Our modifications permit the recovery of and an average of 300 μg RNA per g of needles that is suitable for translationin vitro, northern hybridizations, and the construction of cDNA libraries.     

Development and characterization of 248 novel microsatellite markers in turbot (Scophthalmus maximus).

The turbot is a flatfish species of great relevance to marine aquaculture in Europe. Only a limited number of microsatellites have been isolated to date in this species. To increase the number of potentially useful mapping markers, we screened simple sequence repeat (SSR)--enriched genomic libraries obtained from several di-, tri-, and tetranucleotide tandem repeat motifs. A total of 248 new polymorphic microsatellites were successfully optimized. The efficiency of the protocol applied (6.4%) was higher than that in other studies of fish that used the same method. Dinucleotide and perfect microsatellites were predominant in this species; the (AC)n motif was the most frequent class of repeat. Polymorphism and structural properties at these loci, together with 30 variable loci previously reported in turbot, were evaluated in 6 wild individuals. The number of alleles per locus ranged from 2 to 10, with an average of 4.046. The microsatellite markers characterized in this study will contribute to the development of the turbot genetic map, which can be used for quantitative trait locus (QTL) identification, marker-assisted selection programs, and other applications to improve its culture.     

Microsatellite loci isolation in the endangered Gran Canarian blue chaffinch (Fringilla teydea polatzeki) and their utility in closely related taxa

The blue chaffinch, Fringilla teydea, is an endemic species of the Canary Islands. This species is formed by two subspecies: The Teneriffean blue chaffinch (F. t. teydea), and the endangered Gran Canarian blue chaffinch, (F. t. polatzeki). Here we report the isolation and characterization of nine tetranucleotide microsatellites (AAAG and AAAT) from the Gran Canarian subspecies, using an enrichment protocol. An average of 7.8 alleles per locus and an average observed heterozygosity of 0.773 were found (n = 28). The loci were tested for their ability to cross amplify in the Teneriffean subspecies and in the common chaffinch (Fringilla coelebs). These microsatellites will be used to manage a captive breeding programme for the endangered Gran Canarian subspecies.     

Isolation and characterization of polymorphic microsatellite loci in Castanopsis chinensis Hance (Fagaceae)

Twelve novel microsatellite loci were isolated and characterized from enriched genomic libraries of Castanopsis chinensis. Four previously reported microsatellites from Castanopsis cuspidata were cross-amplified in C. chinensis. Forty-two sample trees from a wild population were tested for polymorphism using a set of the 16 polymorphic microsatellites. The average allele number of these microsatellites was 4.6 per locus, ranging from 2 to 7. The ranges of observed and expected heterozygosity were 0.262–1.000 and 0.238–0.818, respectively. Significant deviations from Hardy–Weinberg equilibrium were detected at five loci and no linkage disequilibrium was observed.     

Development of a genome-wide anchored microsatellite map for common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

A total of 150 microsatellite markers developed for common bean (Phaseolus vulgaris L.) were tested for parental polymorphism and used to determine the positions of 100 genetic loci on an integrated genetic map of the species. The value of these single-copy markers was evident in their ability to link two existing RFLP-based genetic maps with a base map developed for the Mesoamerican × Andean population, DOR364 × G19833. Two types of microsatellites were mapped, based respectively on gene-coding and anonymous genomic-sequences. Gene-based microsatellites proved to be less polymorphic (46.3%) than anonymous genomic microsatellites (64.3%) between the parents of two inter-genepool crosses. The majority of the microsatellites produced single bands and detected single loci, however four of the gene-based and three of the genomic microsatellites produced consistent double or multiple banding patterns and detected more than one locus. Microsatellite loci were found on each of the 11 chromosomes of common bean, the number per chromosome ranging from 5 to 17 with an average of ten microsatellites each. Total map length for the base map was 1,720 cM and the average chromosome length was 156.4 cM, with an average distance between microsatellite loci of 19.5 cM. The development of new microsatellites from sequences in the Genbank database and the implication of these results for genetic mapping, quantitative trait locus analysis and marker-assisted selection in common bean are described.Communicated by H.F. Linskens     

Characterization of (GT)n and (CT)n microsatellites in two insect species: Apis mellifera and Bombus terrestris.

A set of 52 (CT)n and 23 (GT)n microsatellites in honeybee, 24 (CT)n and 2 (GT)n microsatellites in bumble-bee (n > 6) have been isolated from partial genomic libraries and sequenced. On average, (CT)n and (GT)n microsatellites occur every 15 kb and 34 kb in honeybee and every 40 kb and 500 kb in bumble-bee, respectively. The prevailing categories are imperfect repeats for (CT)n microsatellites in bumble-bee, and perfect repeats for both (CT)n and (GT)n microsatellites in honey-bee. Comparisons with data available in vertebrates indicate a lower proportion of perfect repeats in bees but length distributions are very similar regardless the phylum. This result extends to insects the concept of an evolutionary conservation for quantitative and qualitative characteristics of (CT)n and (GT)n microsatellites. Many (CT)n and (GT)n repeats are surrounded with various types of microsatellites, revealing an associative distribution of short repeat sequences. As expected, a high level of intrapopulational polymorphism has been found with one tested honeybee microsatellite. Also, flanking regions of this microsatellite are similar enough to allow PCR amplification in several other species of Apis and Bombus.     

Chicken microsatellite markers isolated from libraries enriched for simple tandem repeats

The total number of microsatellite loci is considered to be at least 10-fold lower in avian species than in mammalian species. Therefore, efficient large-scale cloning of chicken microsatellites, as required for the construction of a high-resolution linkage map, is facilitated by the construction of libraries using an enrichment strategy. In this study, a plasmid library enriched for tandem repeats was constructed from chicken genomic DNA by hybridization selection. Using this technique the proportion of recombinant clones that cross-hybridized to probes containing simple tandem repeats was raised to 16%, compared with < 0·1% in a non-enriched library. Primers were designed from 121 different sequences. Polymerase chain reaction (PCR) analysis of two chicken reference pedigrees enabled 72 loci to be localized within the collaborative chicken genetic map, and at least 30 of the remaining loci have been shown to be informative in these or other crosses.     

The utility of fast evolving molecular markers for studying speciation in the Antarctic benthos

The Southern Ocean is surprisingly rich in species that coexist in one of the most extreme environments on Earth yet the processes leading to speciation in this ecosystem are not well understood. To remedy this, tools that measure the genetic connectedness within a species are needed. Although useful for phylogenetic purposes, the readily available mitochondrial markers (e.g. 16S, COI) suffer from numerous shortcomings for population genetics. Therefore, molecular markers are needed that are sufficiently variable, unlinked, biparentally inherited, and distributed over the whole genome. We argue that microsatellites are suitable markers that have not been widely used in exploratory studies due to their difficult initial set-up. Working with the Ceratoserolis trilobitoides species complex (Isopoda), we demonstrate that using a novel protocol many microsatellites can be identified quickly. An increased availability of these highly sensitive markers will be useful for studies addressing the origin of species in the Southern Ocean and their response to future climate change. Christoph Held and Florian Leese contributed equally to this paper.     

Rapid evolution of mammalian X-linked testis microRNAs

Background

Microsatellites in cDNA are useful as molecular markers because they represent transcribed genes and can be used as anchor markers for linkage and comparative mapping, as well as for studying genome evolution. Microsatellites in cDNA can be detected in existing ESTs by data mining. However, in most fish species, no ESTs are available or the number of ESTs is limited, although fishes represent half of the vertebrates on the earth. We developed a simple and efficient method for isolation of microsatellites from cDNA in fish.

Results

The method included normalization of 150 ng cDNA using 0.5 U duplex-specific nuclease (DSN) at 65°C for 30 min, enrichment of microsatellites using biotinylated oligonucleotides and magnetic field, and directional cloning of cDNA into a vector. We tested this method to enrich CA- and GA-microsatellites from cDNA of Asian seabass, and demonstrated that enrichment of microsatellites from normalized cDNA could increased the efficiency of microsatellite isolation over 30 times as compared to direct sequencing of clones from cDNA libraries. One hundred and thirty-nine (36.2%) out of 384 clones from normalized cDNA contained microsatellites. Unique microsatellite sequences accounted for 23.6% (91/384) of sequenced clones. Sixty microsatellites isolated from cDNA were characterized, and 41 were polymorphic. The average allele number of the 41 microsatellites was 4.85 ± 0.54, while the expected heterozygosity was 0.56 ± 0.03. All the isolated microsatellites inherited in a Mendelian pattern.

Conclusion

Normalization of cDNA substantially increased the efficiency of enrichment of microsatellites from cDNA. The described method for isolation of microsatellites from cDNA has the potential to be applied to a wide range of fish species. The microsatellites isolated from cDNA could be useful for linkage and comparative mapping, as well as for studying genome evolution.     

Spatial and temporal patterns of environmental DNA detection to inform sampling protocols in lentic and lotic systems

The development of efficient sampling protocols for the capture of environmental DNA (eDNA) could greatly help improve accuracy of occupancy monitoring for species that are difficult to detect. However, the process of developing a protocol in situ is complicated for rare species by the fact that animal locations are often unknown. We tested sampling designs in lake and stream systems to determine the most effective eDNA sampling protocols for two rare species: the Sierra Nevada yellow‐legged frog (Rana sierrae) and the foothill yellow‐legged frog (Rana boylii). We varied water volume, spatial sampling, and seasonal timing in lakes and streams; in lakes we also tested multiple filter types. We found that filtering 2 L versus 1 L increased the odds of detection in streams 5.42X (95% CI: 3.2–9.19X) in our protocol, from a probability of 0.51–0.85 per technical replicate. Lake sample volumes were limited by filter clogging, and we found no effect of volume or filter type. Sampling later in the season increased the odds of detection in streams by 1.96X for every 30 days (95% CI: 1.3–2.97X) but there was no effect for lakes. Spatial autocorrelation of the quantity of yellow‐legged frog eDNA captured in streams ceased between 100 and 200 m, indicating that sampling at close intervals is important.     

Isolation and characterization of polymorphic microsatellite markers in Tetranychus urticae and cross amplification in other Tetranychidae and Phytoseiidae species of economic importance

Tetranychus urticae Koch is a cosmopolitan phytophagous mite considered as the most polyphagous species among spider mites. Population genetic studies using molecular markers such as microsatellites have proven to be extremely informative to address questions about population structure, phylogeography and host preferences. The aim of this study was to increase the available molecular tools to gain insight into the genetic structure of T. urticae populations of citrus orchards, which might help in their management. Five microsatellite DNA libraries were developed using probes with the motifs CT, CTT, GT and CAC following the FIASCO protocol. Positive clones, those that included the insert with the microsatellite, were detected using the PIMA-PCR technique. Combinations of primers were designed on 22 out of 32 new microsatellites loci and their polymorphism was tested in four populations sampled along the eastern coast of Spain. Eleven successful amplifications were obtained. Cross amplification was tested in the tetranychids Aphlonobia histricina, Eutetranychus banksi, E. orientalis, Oligonychus perseae, Panonychus citri, Tetranychus evansi, T. okinawanus and T. turkestani, and the phytoseiids Amblyseius swirskii, A. cucumeris, A. andersoni, Euseius stipulatus, Neoseiulus barkeri, N. californicus, Phytoseiulus persimilis and Typhlodromus phialatus. Eight successful cross amplifications were obtained.     

How bryophytes came out of the cold: successful cryopreservation of threatened species

The use of ex situ techniques for the conservation of threatened plants has been increasing over the past decades. Cryopreservation is often used for the long-term storage of plant germplasm where conventional methods (i.e. seedbanking) are inappropriate. Simple encapsulation–dehydration protocols were developed for the cryopreservation of bryophytes at The Royal Botanic Gardens, Kew, as part of an ex situ project for the conservation of UK threatened species. The applicability of these methods was tested on 22 species with a broad range of ecological requirements and found to be highly successful across the board. Regeneration rates from frozen material were >68% for all species tested and half had regeneration rates of 100%. The high regeneration rate and broad applicability of the protocols across a range of species was attributed to a combination of the inherent totipotency of bryophytes and the in-built recovery periods in the pre-treatment protocol. In conclusion, bryophytes are well suited to cryopreservation and such techniques would be applicable for the long-term storage of similar conservation collections across the globe.     

A set of 99 cattle microsatellites: characterization,synteny mapping,and polymorphism

Cattle microsatellite clones (136) were isolated from cosmid (10) and plasmid (126) libraries and sequenced. The dinucleotide repeats were studied in each of these sequences and compared with dinucleotide repeats found in other vertebrate species where information was available. The distribution in cattle was similar to that described for other mammals, such as rat, mouse, pig, or human. A major difference resides in the number of sequences present in the bovine genome, which seemed at best one-third as large as in other species. Oligonucleotide primers (117 pairs) were synthesized, and a PCR product of expected size was obtained for 88 microsatellite sequences (75%). Synteny or chromosome assignment was searched for each locus with PCR amplification on a panel of 36 hamster/bovine somatic cell hybrids. Of our bovine microsatellites, eighty-six could be assigned to synteny groups of chromosomes. In addition, 10 other microsatellites—HEL 5, 6, 9, 11, 12, 13 (Kaukinen and Varvio 1993), HEL 4, 7, 14, 15—as well as the microsatellite found in the -casein gene (Fries et al. 1990) were mapped on the hybrids. Microsatellite polymorphism was checked on at leat 30 unrelated animals of different breeds. Almost all the autosomal and X Chr microsatellites displayed polymorphism, with the number of alleles varying between two and 44. We assume that these microsatellites could be very helpful in the construction of a primary public linkage map of the bovine genome, with an aim of finding markers for Economic Trait Loci (ETL) in cattle.     

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