Cross-amplification and sequence variation of microsatellite loci in Eurasian hard pines

Microsatellite transfer across coniferous species is a valued methodology because de novo development for each species is costly and there are many species with only a limited commodity value. Cross-species amplification of orthologous microsatellite regions provides valuable information on mutational and evolutionary processes affecting these loci. We tested 19 nuclear microsatellite markers from Pinus taeda L. (subsection Australes) and three from P. sylvestris L. (subsection Pinus) on seven Eurasian hard pine species (P. uncinata Ram., P. sylvestris L., P. nigra Arn., P. pinaster Ait., P. halepensis Mill., P. pinea L. and P. canariensis Sm.). Transfer rates to species in subsection Pinus (36–59%) were slightly higher than those to subsections Pineae and Pinaster (32–45%). Half of the trans-specific microsatellites were found to be polymorphic over evolutionary times of approximately 100 million years (ten million generations). Sequencing of three trans-specific microsatellites showed conserved repeat and flanking regions. Both a decrease in the number of perfect repeats in the non-focal species and a polarity for mutation, the latter defined as a higher substitution rate in the flanking sequence regions close to the repeat motifs, were observed in the trans-specific microsatellites. The transfer of microsatellites among hard pine species proved to be useful for obtaining highly polymorphic markers in a wide range of species, thereby providing new tools for population and quantitative genetic studies.Communicated by D.B. NealeS.C. González-Martínez and J.J. Robledo-Arnuncio have contributed equally to this work and are to be considered as joint first authors     

Evolution of a triplet repeat in a conifer.

The opportunity to trace the evolution of a triplet repeat is rare, especially for seed-plant lineages with a well-defined fossil record. Microsatellite PtTX2133 sequences from 18 species in 2 conifer genera were used to calibrate the birth of a CAGn repeat, from its protomicrosatellite origins to its repeat expansion. Birth occurred in the hard-pine genome ~ 136 million years ago, or 14 million generations ago, then expanded as a polymorphic triplet repeat 136-100 million years before a major North American vicariance event. Calibration of the triplet-repeat birth and expansion is supported by the shared allelic lineages among Old and New World hard pines and the shared alleles solely among North American diploxylon or hard pines. Five CAGn repeat units appeared to be the expansion threshold for Old and New World diploxylon pines. Haploxylon pine species worldwide did not undergo birth and repeat expansion, remaining monomorphic, with a single imperfect 198-bp allele. A sister genus, Picea, had only a region of cryptic simplicity, preceding a proto-microsatellite region. The polymorphic triplet repeat in hard pines is older than some long-lived microsatellites reported for reptiles, yet younger than those reported for insects. Some cautionary points are raised about phylogenetic applications for this long-lived microsatellite.     

Rapid expansion of microsatellite sequences in pines

Microsatellite persistence time and evolutionary change was studied among five species of pines, which included a pair of closely related species (Pinus sylvestris and Pinus resinosa) in the subgenus Pinus, their relative Pinus radiata, and another closely related species pair (Pinus strobus and Pinus lambertiana) in the subgenus Strobus. The effective population sizes of these species are known to have ranged from the very small bottlenecks of P. resinosa to vast populations of P. sylvestris. This background allowed us to place the microsatellite evolution in a well-defined phylogenetic setting. Of 30 loci originating from P. strobus and P. radiata, we were able to consistently amplify 4 in most of the these pine species. These priming sites had been conserved for over 100 Myr. The four microsatellites were sequenced in the five species. Flanking sequences were compared to establish that the loci were orthologous. All microsatellites had persisted in these species, despite very different population sizes. We found a recent microsatellite duplication: a closely related pair of loci in P. strobus, where the other four species had just one locus. On two independent occasions, the repeat area of this same microsatellite (locus RPS 105a/b) had grown from a very low repeat number to 15 or 17 in the last 10-25 Myr. Other parts of the same compound microsatellite had remained virtually unchanged. Locus PR 4.6 is known to be polymorphic in both P. radiata and P. sylvestris, but the polymorphism in the two species is due to different motifs. The very large pine genomes are highly repetitive, and microsatellite loci also occur as gene families.     

Transpecific microsatellites for hard pines

Microsatellites are difficult to recover from large plant genomes so cross-specific utilisation is an important source of markers. Fifty microsatellites were tested for cross-specific amplification and polymorphism to two New World hard pine species, slash pine (Pinus elliottii var. elliottii) and Caribbean pine (P. caribaea var. hondurensis). Twenty-nine (58%) markers amplified in both hard pine species, and 23 of these 29 were polymorphic. Soft pine (subgenus Strobus) microsatellite markers did amplify, but none were polymorphic. Pinus elliottii var. elliottii and P. caribaea var. hondurensis showed mutational changes in the flanking regions and the repeat motif that were informative for Pinus spp. phylogenetic relationships. Most allele length variation could be attributed to variability in repeat unit number. There was no evidence for ascertainment bias.     

Low-copy microsatellite markers for Pinus taeda L.

Eighteen low-copy and genomic microsatellite markers were tested for Mendelian inheritance and then assayed in 41 Pinus taeda L. samples drawn from five regions in the southern United States. The PCR products had multiple alleles, high levels of polymorphism, and little non-specific priming. Fifteen of the 18 markers were informative for a P. taeda three-generation RFLP (restriction fragment length polymorphism) pedigree, and a P. taeda population survey revealed three to 28 alleles per locus. The highest allele numbers and polymorphic information content (PIC) values were associated with complex repeat sequences and (or) with sequences consisting of the longer strings of perfect repeats. The abundance of low- to rare-frequency alleles also accounted for high PIC values in both types of markers. Low-copy microsatellites are useful for the large, complex pine genome, especially in the absence of entire gene sequences in public databases and with the low levels of polymorphism in markers developed from expressed sequence tags (ESTs).     

牙鲆CA/GT微卫星标记的筛选

采用生物素选择杂交法与放射性同位素杂交法相结合的技术,成功地从牙鲆(Paralichthys olivaceus)基因组中分离出含有CA/GT重复类型的微卫星序列。通过两轮淘选,共获得526个阳性菌落。测序其中的119个菌落,结果获得133个含有微卫星座位的序列。除了两个复合型微卫星外(1.5%),完美型63个(47.37%),非完美型68个(51.13%)。设计并合成22对微卫星引物,对8个人工雌核发育家系的亲本进行遗传背景分析。PCR结果表明,4对引物无扩增带或者扩增带不是目的条带,1对引物表现为单态,其余17对引物均呈多态性,平均每个座位产生5.2个复等位基因,杂合度为0.375～0.846,多态信息含量为0.305～0.823。结果表明,所筛选的大部分微卫星标记能够用于牙鲆群体遗传学研究。     

Characterization and isolation of novel microsatellites from the Drosophila dunni subgroup

We have isolated and characterized 77 novel microsatellites from two species, Drosophila dunni and Drosophila nigrodunni, which are closely related Caribbean-island endemics from the Drosophila cardini species group. These species are very distantly related to all other Drosophila from which microsatellites have previously been characterized. We find that the average length of microsatellites isolated in these species is quite small, with an overall mean length of 9.8 repeat units for dinucleotide microsatellites in the two study species. The nucleotide composition of dinucleotides differs between the two species: D. nigrodunni has a predominance of (AC/GT)n repeats, whereas D. dunni has equal numbers of (AC/GT)n and (AG/CT)n repeats. Tri- and tetranucleotide repeats are not abundant in either species. We assayed the variability of eight microsatellites in a closely related third species, Drosophila arawakana, using wild-caught individuals from the island of Guadeloupe. We found the microsatellites to be extremely variable in this population, with observed heterozygosities ranging from 0.541 to 0.889. DNA amplification trials suggest that these eight microsatellites are widely conserved across the D. cardini group, with five of the eight producing amplification products in every species tested. However, the loci are very poorly conserved over greater phylogenetic distances. DNA amplification of the microsatellite loci was unreliable in members of the closely related Drosophila quinaria, Drosophila calloptera, Drosophila guarani and Drosophila tripunctata species groups. Furthermore, these microsatellites could not be detected in the genome of Drosophila melanogaster, despite the conservation of microsatellite flanking regions at some loci. These data indicate that Drosophila microsatellite loci are quite short lived over evolutionary timescales relative to many other taxa.     

Suitability of some southern and western pines as hosts for the pine shoot beetle, Tomicus piniperda (Coleoptera: Scolytidae)

The pine shoot beetle, Tomicus piniperda (L.), is an exotic pest that has become established in North America. Discovered in Ohio in 1992, it has since been found in at least 13 states and parts of Canada. The beetle can cause significant growth loss in pines, and it represents a potential threat to trees in areas where it has not yet become established. To evaluate this threat to native pines, field and laboratory tests were conducted on several common and important southern and western species to determine whether they are acceptable hosts for T. piniperda. Comparisons with Pinus sylvestris L., Scots pine, a preferred natural host for the beetle, were made where possible. Measurements of beetle attack success on southern pine billets showed that Pinus taeda L., Pinus echinata Miller, Pinus elliottii var. elliottii Engelmann, Pinus palustris Miller, and Pinus virginiana Miller (loblolly, shortleaf, slash, longleaf, and Virginia pine, respectively) and two western pines, Pinus ponderosa Lawson and Pinus contorta Douglas (ponderosa and lodgepole pine, respectively), were acceptable for breeding material, but brood production was highly variable. Among the southern pines, P. taeda and P. echinata were susceptible to shoot feeding by T. piniperda, whereas P. elliottii was highly resistant and P. palustris seemed to be virtually immune. Shoot feeding tests on the western pines were conducted only in the laboratory, but there was moderate-to-good survival of adults feeding on both species. It seems that if T. piniperda is introduced into the south and west it will likely establish and may cause some damage to native pines. P. taeda may be affected more than other southern pines because it is the most abundant species, it is readily attacked for brood production, which can result in moderately large broods, and the beetle survives well during maturation feeding on P. taeda shoots.     

Extracting DNA from submerged pine wood.

A DNA extraction protocol for submerged pine logs was developed with the following properties: (i) high molecular weight DNA, (ii) PCR amplification of chloroplast and nuclear sequences, and (iii) high sequence homology to voucher pine specimens. The DNA extraction protocol was modified from a cetyltrimehtylammonium bromide (CTAB) protocol by adding stringent electrophoretic purification, proteinase K, RNAse, polyvinyl pyrrolidone (PVP), and Gene Releaser. Chloroplast rbcL (ribulose-1,5-bisphosphate carboxylase) could be amplified. Nuclear ribosomal sequences had >95% homology to Pinus taeda and Pinus palustris. Microsatellite polymorphism for PtTX2082 matched 2 of 14 known P. taeda alleles. Our results show DNA analysis for submerged conifer wood is feasible.     

Development and characterization of SSR markers in Ribes species

Eleven microsatellite loci were identified and characterized in blackcurrant (Ribes nigrum L.) and related species. An enriched library was constructed and screened with simple sequence repeat (SSR) oligonucleotides. Positive clones were sequenced and primers were designed flanking the repeat motifs. These 11 microsatellites produce amplification products polymorphic across a range of Ribes germplasm, predominantly from the Eucoreosma section of the genus. The number of alleles varied from 2 to 18 with levels of diversity ranging from 0.18 to 0.91.     

Sequence-tagged site markers for microsatellites: Simplified technique for rapidly obtaining flanking sequences

A sequencing strategy is described for the rapid recovery of DNA sequences flanking repeat sequences of microsatellites in plant nuclear genomes. Primers that represent a perfect microsatellite repeat and end in a 3′ degenerate base have been used to sequence directly from microsatellite repeats in one direction. The procedure allows the design of one flanking primer that is then used to sequence back through the repeat to define the microsatellite site precisely and also provides for the design of the second flanking primer. The strategy is versatile with various repeat sizes and different categories of microsatellites; perfect, imperfect, and compound were found to be suitable templates for analysis.     

Isolation of highly polymorphic microsatellite loci for a species with a large genome size: sharp-ribbed salamander (Pleurodeles waltl)

Fourteen highly polymorphic microsatellite markers were developed and characterized for the sharp-ribbed salamander, Pleurodeles waltl. Isolating microsatellites with more than 12 single repeat type units was only successful for a tetranucleotide repeat (ATAG). Compared to microsatellite libraries constructed simultaneously for two anuran amphibian species, a greater number of primer pairs designed for P. waltl had to be discarded, due to consistent amplification problems. Low amplification success rate for P. waltl may be due to its larger genome size. Consequently, to avoid nonspecific binding and to increase amplification success, polymerase chain reaction programmes with touchdown cycles were used. For 14 microsatellite markers, amplification was successful and consistent with number of alleles and expected heterozygosity ranging from seven to 22 and from 0.79 to 0.94, respectively. All 14 microsatellite markers will be extremely useful for metapopulation studies of this unique amphibian species.     

In silico whole-genome EST analysis reveals 2322 novel microsatellites for the silver-lipped pearl oyster, Pinctada maxima

Molecular stock improvement techniques such as marker assisted selection have great potential in accelerating selective breeding programmes for animal production industries. However, the discovery and application of trait/marker associations usually requires a large number of genome-wide polymorphic loci. Here, we present 2322 unique microsatellites for the silver-lipped pearl oyster, Pinctada maxima, a species of aquaculture importance throughout the Indo-Australian Archipelago for production of the highly valued South Sea pearl. More than 1.2 million Roche 454 expressed sequence tag (EST) reads were screened for microsatellite repeat motifs. A total of 12,604 sequences contained either a di, tri, tetra, penta or hexa microsatellite repeat motif (n ≥ 6), with 6435 of these sequences having sufficient flanking regions for primer development. All identified microsatellites with designed primers were condensed into 2322 unique clusters (i.e., unique loci) of which 360 were shown to be polymorphic based on multiple sequence reads with different repeat motifs. Genotyping of five microsatellite loci demonstrated that in silico evaluation of polymorphism levels was a very useful method for identification of polymorphic loci, with the variation uncovered being a lower bound. Gene Ontology annotations of sequences containing microsatellites suggest that most are derived from a diverse array of unique genes. This EST derived microsatellite database will be a valuable resource for future studies in genetic map construction, diversity analysis, quantitative trait loci analysis, association mapping and marker assisted selection, not only for P. maxima, but also closely related species within the genus Pinctada.     

Comparative analysis of microsatellite loci in four fruit fly species of the genus Ceratitis (Diptera: Tephritidae)

The possibility to cross-species amplify microsatellites in fruit flies of the genus Ceratitis was tested with the polymerase chain reaction (PCR) by analysing 23 Ceratitis capitata (Wiedemann) microsatellite markers on the genomic DNA of three other economically important, congeneric species: C. rosa (Karsch), C. fasciventris (Bezzi) and C. cosyra (Walker). Twenty-two primer pairs produced amplification products in at least one of the three species tested. The majority of the products were similar, if not identical in size to those expected in C. capitata. The structures of the repeat motifs and their flanking sequences were examined for a total of 79 alleles from the three species. Sequence analysis revealed the same repeat type as the homologous C. capitata microsatellites in the majority of the loci, suggesting their utility for population analysis across the species range. A total of seven loci were differentially present/absent in C. capitata, C. rosa, C. fasciventris and C. cosyra, suggesting that it may be possible to differentiate these four species using a simple sequence repeat-based PCR assay. It is proposed that medfly-based microsatellite markers could be utilized in the identification and tracing of the geographical origins of colonist pest populations of the four tested species and in the assessment of their risk and invasive potentials; thereby assisting regulatory authorities in implementing quarantine restrictions and other pest control measures.     

Families of clustered microsatellites in a conifer genome

Microsatellite clustering may account for genetic maps which do not coalesce into the expected number of linkage groups. Microsatellite organization within the large genome of Pinus taeda (1C = 20,000 Mb) was determined by (1) testing whether repeat motifs were sequestered within the low-copy DNA kinetic component and (2) testing for repeat motif clusters within DNA fragments regardless of copy number. Within the low-copy kinetic component, either (AC)n or (AG)n repeat units were present in 32% of sequences. No repeat motifs were found in the total genome control. Clustered repeat motifs were frequent; the (ATG)n triplet repeat motif was located upstream from a CG-rich trinucleotide microsatellite in 26 out of 44 microsatellite sequences. Fourteen of the clustered (ATG)n sequences could be assembled into four microsatellite sequence families based on similarities in the flanking regions. Consistent with the DNA turnover model, family members shared similar flanking regions but differed in repeat motif composition and length.     

Microsatellite marker identification using genome screening and restriction-ligation

We have identified a fast and easy method for finding microsatellite markers that utilizes genome screening with inter-simple sequence repeat (ISSR) primers to detect microsatellite regions and to obtain sequence information flanking one side of the microsatellites and a restriction-ligation technique with a specific adaptor to allow sequence walking to obtain sequence information flanking the other side of the microsatellites. Two main alternatives of the method (with or without cloning) are presented. We successfully utilized the method when identifying microsatellite markers for 21 bryophyte species, three algal species, and for the raccoon dog. The proportion of polymorphic markers equaled 95%. We observed that microsatellites are commonly found within the sequenced ISSR amplification products (54% in the present study), in which case specific primers can be identified for the microsatellite without a further restriction-ligation step. It is evident that the DNA regions amplified by ISSR markers commonly represent microsatellite hotspots. We propose that the identified method and the knowledge of the common presence of additional microsatellite repeats within ISSR amplification products are especially attractive to researchers who conduct small-scale microsatellite identification, such as researchers in population genetics and conservation biology.     

Evidence for complex mutations at microsatellite loci in Drosophila.

Fifteen lines each of Drosophila melanogaster, D. simulans, and D. sechellia were scored for 19 microsatellite loci. One to four alleles of each locus in each species were sequenced, and microsatellite variability was compared with sequence structure. Only 7 loci had their size variation among species consistent with the occurrence of strictly stepwise mutations in the repeat array, the others showing extensive variability in the flanking region compared to that within the microsatellite itself. Polymorphisms apparently resulting from complex nonstepwise mutations involving the microsatellite were also observed, both within and between species. Maximum number of perfect repeats and variance of repeat count were found to be strongly correlated in microsatellites showing an apparently stepwise mutation pattern. These data indicate that many microsatellite mutation events are more complex than represented even by generalized stepwise mutation models. Care should therefore be taken in inferring population or phylogenetic relationships from microsatellite size data alone. The analysis also indicates, however, that evaluation of sequence structure may allow selection of microsatellites that more closely match the assumptions of stepwise models.     

Conservation of a dinucleotide simple sequence repeat locus in sharks

Recent studies indicate that the flanking region and repeat motif structure of conserved microsatellite loci are useful for phylogenetic inference. Most comparative studies of microsatellite loci involve relatively closely related species, however, primarily because primers developed for one species often amplify only related species. We describe an analysis of a microsatellite locus in lamniform sharks that we estimate has been conserved for a billion years. Combined analysis of the flanking sequence and repeat motif structure resulted in a gene tree comparable to those reported from similar analyses of other genes. The conservation of the simple sequence repeat (SSR), and of the sequence flanking the SSR, is explained by a low substitution rate in sharks coupled with the possibility that mutations which interrupt perfect repeats are lost by replication slippage.     

Isolation and characterization of microsatellites in lane snapper (Lutjanus synagris), mutton snapper (Lutjanus analis), and yellowtail snapper (Ocyurus chrysurus)

Polymerase chain reaction primer pairs for a total of 25 nuclear‐encoded microsatellites (loci) were developed from genomic DNA libraries of lane snapper (Lutjanus synagris), mutton snapper (Lutjanus analis), and yellowtail snapper (Ocyurus chrysurus). The microsatellites include 24 perfect (21 dinucleotide and three trinucleotide) and one imperfect (combination tetranucleotide/tetranucleotide) repeat motifs. A total of 32 individuals of each species were assayed for allelic variation at all 25 microsatellites; reliable amplification products were generated for lane snapper (25 loci), mutton snapper (21 loci), and yellowtail snapper (24 loci). Significant deviations from Hardy–Weinberg expectations, following Bonferroni corrections, were found for one microsatellite in lane and yellowtail snappers, and for three microsatellites in mutton snapper. All pairwise comparisons of microsatellites (all three species) did not deviate significantly from genotypic equilibrium.     

Complex evolution of a highly conserved microsatellite locus in several fish species

The evolutionary dynamics of a highly conserved microsatellite locus ( Dla 11 ) were studied in several fish species. The data indicated that multiple types of compound microsatellites arose through point mutations that were sometimes followed by expansion of the derived motif. Furthermore, extensive length variation was detected among species in the regions immediately flanking the repeat region.