An integrated genetic and cytogenetic map for the Mediterranean fruit fly, <Emphasis Type="Italic">Ceratitis capitata</Emphasis>, based on microsatellite and morphological markers

A genetic map based on microsatellite polymorphisms and visible mutations of the Mediterranean fruit fly (medfly), Ceratitis capitata is presented. Genotyping was performed on single flies from several backcross families. The map is composed of 67 microsatellites and 16 visible markers distributed over four linkage groups. Fluorescence in situ hybridization of selected microsatellite markers on salivary gland polytene chromosomes allowed the alignment of these groups to the second, fourth, fifth and sixth chromosome. None of the markers tested showed segregation either with the X or the third chromosome. However, this map constitutes a substantial starting point for a detailed genetic map of C. capitata. The construction of an integrated map covering the whole genome should greatly facilitate genetic studies and future genome sequence projects of the species.     

A convenient protocol for extraction and purification of DNA from Fragaria

Summary In this paper we describe a simple and efficient DNA extraction protocol for Fragaria species, a highly recalcitrant genus due to the large amount of polyphenols and polymeric carbohydrates present in strawberry tissues. The protocol yields a high quality DNA that can be amplified by polymerase chain reaction and digested with restriction endonucleases.     

High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae)

The objectives of the present study were to develop microsatellite markers for the wild strawberry, Fragaria virginiana, to evaluate segregation patterns of microsatellite alleles in this octoploid species, and assess genetic variability at microsatellite loci in a wild population. A genomic library was screened for microsatellite repeats and several PCR primers were designed and tested. We also tested the use of heterologous primers and found that F. virginiana primers amplified products in cultivated strawberry, Fragaria × ananassa Duch. and Fragaria chiloensis. Similarly, microsatellite loci developed from cultivated strawberry also successfully amplified F. virginiana loci. We investigated four microsatellite loci in detail, three developed from F. virginiana and one from cultivated strawberry. A survey of 100 individuals from a population of F. virginiana in Pennsylvania demonstrated high heterozygosities (H_e or gene diversity ranged from 0.80 to 0.88 per locus) and allelic diversity (12–17 alleles per locus), but individual plants had no more than two alleles per locus. Segregation patterns in parents and progeny of two controlled crosses at these four loci were consistent with disomic Mendelian inheritance. Together these findings suggest that the genome of F. virginiana is "highly diploidized" and at least a subset of microsatellite loci can be treated as codominant, diploid markers. Significant heterozygote deficiencies were found at three of the four loci for hermaphroditic individuals but for only one locus among females in this gynodioecious species.Communicated by J. Dvorak     

Investigation of mutant frequency at the HPRT locus and changes in microsatellite sequences in healthy young adults

In an attempt to understand the inter-individual variation that occurs in in vivo mutant frequency at the HPRT locus, we have examined the effect of polymorphisms in genes for metabolic enzymes on the mutation rate. In the same population of human volunteers, the background variant frequency in a number of microsatellite sequences was studied to determine individual variation in the capacity to repair mismatches in these sequences. The HPRT mutant frequency of T-cells isolated from a group of 49 healthy, non-smoking adults varied from 0.25 to 9.64×10⁻⁶. The frequency of polymorphisms in CYP1A1, GSTM1 and NAT2 among these individuals was similar to those published, and when subjected to univariate analysis these polymorphisms showed no influence on the HPRT mutant frequency. However, there was a significant interaction between the GSTM1 null genotype and the slow acetylator status in NAT2 (P<0.05) which was associated with higher mutant frequency. Analysis of 30 microsatellite sequences in 20 HPRT proficient clones per individual showed only six alterations in total, giving an overall mutation rate per allele of 0.01%, whilst three alterations were found in five HPRT deficient clones per individual examined for changes in 10 microsatellites, giving an overall mutation rate per allele of 0.3%. Thus, the alterations detected are probably due to background mutations and not to differences in mismatch repair capacity.     

Isolation and characterization of 12 microsatellite loci from cutlassfish (Trichiurus haumela)

The cutlassfish (Trichiurus haumela) is an important commercial fish species. In the present study, we report 12 polymorphic microsatellite DNA markers for cutlassfish. The number of alleles per locus ranged from 2 to 9 in a sample of 26 individuals. Observed and expected heterozygosities per locus varied from 0.2727 to 0.9583 and from 0.4059 to 0.7926, respectively. Two loci (Trha25 and Trha27) showed significant departure from Hardy–Weinberg equilibrium after sequential Bonferroni correction (P < 0.0042). No significant linkage disequilibrium between pairs of loci was found. These microsatellite markers provide powerful tools for investigating genetic population structure, population history and conservation management of cutlassfish. Jin-Zhen Bi and Chang-Wei Shao contributed equally to this work.     

A genetic map of 1,000 SSR and DArT markers in a wide barley cross

A high-density genetic map was developed from an F1-derived doubled haploid population generated from a cross between cultivated barley (Hordeum vulgare) and the subspecies H. vulgare ssp. spontaneum. The map comprises 1,000 loci, amplified using 536 SSR (558 loci) and 442 DArT markers. Of the SSRs, 149 markers (153 loci) were derived from barley ESTs, and 7 from wheat ESTs. A high level of polymorphism (∼70%) was observed, which facilitated the mapping of 197 SSRs for which genetic assignments had not been previously reported. Comparison with a published composite map showed a high level of co-linearity and telomeric coverage on all seven chromosomes. This map provides access to previously unmapped SSRs, improved genome coverage due to the integration of DArT and EST-SSRs and overcomes locus order issues of composite maps constructed from the alignment of several genetic maps. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An RFLP linkage map for loblolly pine based on a three-generation outbred pedigree

A genetic linkage map for loblolly pine (Pinus taeda L.) was constructed using segregation data from a three-generation outbred pedigree consisting of four grandparents, two parents, and 95 F₂ progeny. The map was based predominantly on restriction fragment length polymorphism (RFLP) loci detected by cDNA probes. Sixty-five cDNA and three genomic DNA probes revealed 90 RFLP loci. Six polymorphic isozyme loci were also scored. One-fourth (24%) of the cDNA probes detected more than 1 segregating locus, an indication that multigene families are common in pines. As many as six alleles were observed at a single segregating locus among grandparents and it was not unusual for the progeny to segregate for three or four alleles per locus. Multipoint linkage analysis placed 73 RFLP and 2 isozyme loci into 20 linkage groups; the remaining 17 RFLP and 4 isozyme loci were unlinked. The mapped RFLP probes provide a new set of codominant markers for genetic analyses in loblolly pine.     

Isolation and characterization of polymorphic microsatellite loci in a traditional Chinese medicinal plant, Schisandra sphenanthera

Schisandra sphenanthera (Schisandraceae) is a traditional Chinese medicinal plant. In this study, nine polymorphic microsatellite loci were developed and characterized for the species. A genomic DNA enrichment protocol was used to isolate microsatellite loci and polymorphism was explored using 36 individuals sampled from a natural population. The observed number of alleles ranged from 2 to 10 with an average of 5.2. Expected and observed heterozygosity ranged from 0.30 to 0.82 and 0.33 to 0.97, respectively. These microsatellites have been directly applied to both population and conservation genetic studies of S. sphenanthera.     

Resource sharing among ramets in the clonal herb,Fragaria chiloensis

Summary The herbaceous perennial, Fragaria chiloensis, reproduces vegetatively on coastal sand dunes in California by growth of stolons that bear rosettes. Movement of water and photosynthates through stolons integrates water and carbon metabolism of rosettes both before and after they root. New, unrooted rosettes import sufficient water and nitrogen to maintain levels near those of established rosettes; yet support of an unrooted rosette did not decrease growth of a connected, rooted sibling given abundant light, water, and soil nutrients. Under such conditions strings of unrooted rosettes with the associated stolon appeared self-sufficient for carbon; shade and drought induced import of photosynthates. New rosettes produced and maintained a limited root mass upon contact with dry sand, which could increase probability of establishment. Rooting did not induce senescence of stolons. Connection between two established rosettes prevented death by drought and shade, even when neither rosette could have survived singly. Results suggest that physiological integration of connected rosettes may increase total growth of clones of F. chiloensis through sharing of resources among ramets, especially when resource availability is changeable or patchy.     

Characterization of 22 microsatellite marker loci in the Madagascar rousette (Rousettus madagascariensis)

Twenty-two nuclear microsatellite loci were isolated from a genomic DNA library derived from Madagascar’s Rousettus madagascariensis. Marker characteristics were determined from a single population (37 individuals) from Fort Dauphin (southeastern Madagascar). Sixteen of the 22 loci were within Hardy–Weinberg expectations. These loci are highly informative with polymorphic information content values ranging between 0.757 and 0.916. These loci will provide valuable information for the study of population genetics and gene flow within this species of bats. Due to the dramatic reduction and alteration of their habitat, data generated utilizing this marker suite will potentially provide additional information for the effective long-term management of this near-threatened bat species.     

Twelve polymorphic microsatellite loci for <Emphasis Type="Italic">Achnatherum inebrians</Emphasis> (Poaceae)

Microsatellites are often highly variable and abundant in most complex genomes, therefore are widely used in population genetic studies. In this study, twelve polymorphic microsatellite loci were isolated and characterized for the Achnatherum inebrians, a plant abundant in grasslands of Northwest China. Characterization of 24 A. inebrians individuals form four geographically distant populations (Gansu, Qinghai, Xinjiang and Inner Mongolia provinces) showed moderate to high allelic diversity ranging from 3 to 13 alleles per locus, and the expected heterozygosity ranging from 0.41 to 0.67. No evidence of linkage disequilibrium was found for any locus pairwise comparisons. The markers described here will be useful for investigating the genetic diversity, genetic structure and gene flow of this species. Na Chen and Yan-Zhuo Yang contributed equally to this work.     

Development of an RFLP linkage map in diploid peanut species

An RFLP linkage map of peanut has been developed for use in genetic studies and breeding programs aimed at improving the cultivated species (Arachis hypogaea L.). An F₂ population derived from the interspecific hybridization of two related diploid species in the sectionArachis (A. stenosperma ×A. cardenasii) was used to construct the map. Both random genomic and cDNA clones were used to develop the framework of the map. In addition, three cDNA clones representing genes coding for enzymes involved in the lipid biosynthesis pathway have been mapped in peanut. Of the 100 genomic and 300 cDNA clones evaluated, 15 and 190, respectively, revealed polymorphisms among the parents of our mapping population. Unfortunately, a large number of these produced complex banding patterns that could not be mapped. Of the 132 markers analyzed for segregation, 117 are distributed among 11 linkage groups, while 15 have not yet been associated with any other marker. A total map distance of approximately 1063 cM has been covered to-date.     

Isolation and characterization of microsatellite loci in Acer opalus (Aceraceae), a sexually-polymorphic tree, through an enriched genomic library

An enrichment protocol was used to isolate and characterize microsatellite loci in Acer opalus, a Mediterranean tree species. Highly polymorphic microsatellite loci were required for paternity analyses in a population of this species. Eight microsatellite loci were amplified and a total of 87 alleles were detected in a sample of 142 individuals from one population, allowing the identification of each individual with a unique multilocus genotype. The paternity exclusion probabilities varied from 0.261 for locus Aop820 to 0.806 for locus Aop450, and the parent-pair exclusion probabilities varied from 0.433 for Aop820 to 0.940 for Aop450. The cumulative probabilities of exclusion for paternity and parentage of the eight loci were both higher than 0.999, supporting the usefulness of these microsatellite loci for future paternity and parentage analyses in A. opalus. Cross-species transferability was also assayed, supporting their potential use in other eight Acer species.     

Characterization of 21 microsatellite marker loci in the silky sifaka (Propithecus candidus)

The silky sifaka, Propithecus candidus, considered one of the rarest and most endangered primates in the world, exists in only a few fragmented forests in northeastern Madagascar. This species faces increasing pressures as a direct result from loss of habitat in the form of tavy (slash and burn agriculture), illegal logging and mining along with hunting for subsistence, even within protected areas. We report a marker suite of 21 loci developed from genomic DNA from a silky sifaka collected in 2003 from Anjanaharibe-Sud Special Reserve. Polymorphism of each locus evaluated in 18 individuals pooled from Marojejy National Park and Anjanaharibe-Sud Special Reserve. The number of observed alleles per locus ranges from 2 to 7. The observed and expected heterozygosity were 0.389–0.889 and 0.322–0.789, respectively. The information revealed in this study will provide useful tools for further study of the social structure and population dynamics of the silky sifaka to facilitate conservation management in the imminent future.     

Isolation and characterization of polymorphic microsatellite loci in <Emphasis Type="Italic">Achnatherum sibiricum</Emphasis> (Poaceae)

Achnatherum sibiricum is a threaten and toxic perennial bunchgrass mainly in the north of China. We developed 11 polymorphic microsatellite loci from A. sibiricum by combining biotin capture method. After validating and scoring, these loci were polymorphic in a test population with the range of alleles from four to 13 per locus. The observed and expected heterozygosities ranged from 0.1649 to 0.5624 and from 0.3071 to 0.8826, respectively. All 11 microsatellite markers were in Hardy–Weinberg equilibrium. These polymorphic microsatellites will be useful for genetic diversity analysis and population structure construction for A. sibiricum.     

Isolation via enrichment and characterization of 14 dinucleotide microsatellite loci in one catfish, Glyptosternum maculatum and cross-amplification in other related taxa

A total of 14 novel dinucleotide microsatellite loci were developed using genomic DNA enrichment protocol and characterized for one Glyptosternoid catfish, Glyptosternum maculatum. Polymorphism was explored using 36 individuals from one natural population. The observed number of alleles ranged from 2 to 9. The ranges of observed and expected heterozygosity were 0.0278 to 1 and 0.4319 to 0.8498, respectively and the average of PIC was 0.5754. In addition, successful cross-species amplification of these set of microsatellite markers in the related taxa, Pseudechenneis sulcatus (McClelland) suggested that they would provide a useful tool for the genetic and conservation studies of Sisoridae species.     

Development of 15 polymorphic microsatellite loci from <Emphasis Type="Italic">Garrulax morrisonianus</Emphasis> (Timaliidae), an endemic avian species of Taiwan

Garrulax morrisonianus, an endemic avian species of Taiwan, inhabits evergreen forests at high elevations of 2,000–3,952 m. In this study, we developed 15 microsatellite primer pairs for genetic study. These markers were screened for 52 samples collected from wild populations of different geographical regions. The number of alleles ranged from 4 to 13. The expected (H _E) and observed (H _O) heterozygosities were 0.430–0.725 and 0.000–0.500, respectively. All loci were significantly deviated from Hardy-Weinberg equilibrium due to the heterozygote deficiency. The authors Tzen-Yuh Chiang, Sao-Cheng Lao, Ya-Fu Lee contributed to the study equally.     

Development of 12 novel microsatellite loci in a traditional Chinese medicinal plant, Epimedium brevicornum and cross-amplification in other related taxa

A total of 12 polymorphic microsatellite loci were developed and characterized for a Chinese medicinal plant, Epimedium brevicornum (Berberidaceae). A genomic DNA enrichment protocol was used to isolate microsatellite loci and polymorphism was explored using 38 individuals from one natural population. The observed number of alleles ranged from 2–14. The ranges of observed and expected heterozygosity were 0.00–0.83 and 0.15–0.88, respectively. In addition, successful cross-species amplification of this set of microsatellite markers in other four medicinal Epimedium species suggested that they would provide a useful tool for the genetic and conservation studies of Epimedium species.     

A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 11 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.     

Development of a new SSR-based linkage map in apricot and analysis of synteny with existing Prunus maps

Linkage maps of the apricot accessions ‘Lito’ and ‘BO 81604311’ were constructed using a total of 185 simple sequence repeat (SSR) markers sampled from those isolated in peach, almond, apricot and cherry; 74 were derived from a new apricot genomic library enriched for AG/CT microsatellite repeats (UDAp series), and in total, 98 had never been mapped in Prunus before. Eight linkage groups putatively corresponding to the eight haploid apricot chromosomes were identified for each parent. The two maps were 504 and 620 cM long, respectively, with 96 anchor markers showing a complete co-linearity between the two genomes. As few as three gaps larger than 15 cM were present in ‘Lito’ and six in the male parent; the maps align well with all the available SSR-based Prunus maps through the many common anchor loci. Only occasionally inverted positions between adjacent markers were found, and this can be explained by the small size of cross populations analysed in these Prunus maps and in those reported in literature. The newly developed apricot SSRs will help saturating the existing Prunus maps and will extend the choice of markers in the development of genetic maps for new breeding populations.