Inbreeding variability and population structure in the invasive haplodiploid palm‐seed borer (Coccotrypes dactyliperda)

We investigated the mating system and population genetic structure of the invasive haplodiploid palm‐seed borer Coccotrypes dactyliperda in California. We focused on whether these primarily inbreeding beetles have a ‘mixed‐breeding’ system that includes occasional outbreeding, and whether local inbreeding coefficients (F_IS) varied with dominant environmental factors. We also analysed the genetic structure of C. dactyliperda populations across local and regional scales. Based on the analysis of genetic variation at seven microsatellite loci in 1034 individual beetles from 59 populations, we found both high rates of inbreeding and plentiful evidence of mixed‐breeding. F_IS ranged from ?0.56 to 0.90, the highest variability reported within any animal species. There was a negative correlation between F_IS and latitude, suggesting that some latitude‐associated factor affecting mating decisions influenced inbreeding rates. Multiple regressions suggested that precipitation, but not temperature, may be an important correlate. Finally, we found highly significant genetic differentiation among sites, even over short geographic distances (< 1000 m).     

Mate availability contributes to maintain the mixed‐mating system in a scolytid beetle

We investigated the mating system and population genetic structure of the beetle, Coccotrypes dactyliperda, with life history characteristics that suggest the presence of a stable mixed‐mating system. We examined the genetic structure of seven populations in Israel and found significant departures from the Hardy–Weinberg equilibrium and an excess of homozygosity. Inbreeding coefficients were highly variable across populations, suggesting that low levels of outbreeding occur in nature. Experiments were conducted to determine whether the observed high inbreeding in these populations is the result of a reproductive assurance strategy. Females reared in the laboratory took longer to mate with males from the same population (inbreeding) than with males from a different population (outbreeding). These results suggest that females delayed inbreeding, and were more inclined to outbreed when possible. Thus inbreeding, which predominates in most populations, may be due to a shortage of mates for outbreeding rather than a preference for inbreeding. We conclude that C. dactyliperda has a mixed‐mating system that may be maintained by a reproductive assurance strategy.     

Development of nuclear microsatellite markers for the Japanese conifers Tsuga diversifolia and T. sieboldii (Pinaceae)

Nuclear microsatellite markers were developed for the two Tsuga species native to the Japanese Archipelago, Tsuga diversifolia and T. sieboldii, and a population with genetic affinities to T. diversifolia on Ulleung Island, Korea. Tsuga diversifolia and T. sieboldii are widespread dominant trees of temperate and subalpine forests in Japan but to date no genetic markers have been developed for these species. Fifteen polymorphic loci were developed and characterized, of which 14 are reliably amplified in each taxon. Across both species and the Ulleung Island population, the number of alleles per locus ranged from 3 to 26 (average = 13.93) and observed heterozygosity ranged from 0.005 to 0.935 (average = 0.535). In addition, all 15 loci were successfully amplified in a single accession of the Chinese species, T. chinensis. These markers will be useful for investigating the species’ biogeography, range‐wide genetic diversity, conservation genetic issues and potential for hybridisation.     

Development of microsatellite markers for the red tide‐forming harmful species Chattonella antiqua,C. marina,and C. ovata (Raphidophyceae)

We have developed 11 microsatellite markers that are specific to Chattonella antiqua, C. marina, and C. ovata, the red tide‐forming harmful phytoplanktons. The 11 loci were amplified in the three species. The number of alleles per locus ranged from 5 to 16. The three species shared most microsatellite regions, although the genetic differences in specific loci were detected among them. These markers of the Chattonella species will be beneficial for biogeographical, detailed taxonomic, studies.     

Microsatellite loci for euglossine bees (Hymenoptera: Apidae)

The Neotropical Euglossini (Hymenoptera: Apidae) are important pollinators of many flowering plants, particularly orchids. Lack of highly polymorphic genetic markers for euglossine species has limited the study of their social organization and inbreeding. We therefore developed microsatellite markers for two species, Eulaema nigrita (11 loci) and Euglossa cordata (nine loci), most of which were highly polymorphic in the source species and in a range of related euglossine bees.     

A consensus map for <Emphasis Type="Italic">Cucurbita pepo</Emphasis>

Using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), and morphological traits, the first genetic maps for Cucurbita pepo (2n=2x=40) were constructed and compared. The two mapping populations consisted of 92 F₂ individuals each. One map was developed from a cross between an oil-seed pumpkin breeding line and a zucchini accession, into which genes for resistance to Zucchini Yellow Mosaic Virus (ZYMV) from a related species, C. moschata, had been introgressed. The other map was developed from a cross between an oil-seed pumpkin and a crookneck variety. A total of 332 and 323 markers were mapped in the two populations. Markers were distributed in each map over 21 linkage groups and covered an average of 2,200 cM of the C. pepo genome. The two maps had 62 loci in common, which enabled identification of 14 homologous linkage groups. Polyacrylamide gel analyses allowed detection of a high number of markers suitable for mapping, 10% of which were co-dominant RAPD loci. In the Pumpkin-Zucchini population, bulked segregant analysis (BSA) identified seven markers less than 7 cM distant from the locus n, affecting lignification of the seed coat. One of these markers, linked to the recessive hull-less allele (AW11-420), was also found in the Pumpkin-Crookneck population, 4 cM from n. In the Pumpkin-Zucchini population, 24 RAPD markers, previously introduced into C. pepo from C. moschata, were mapped in two linkage groups (13 and 11 markers in LGpz1 and LGpz2, respectively), together with two sequence characterized amplified region (SCAR) markers linked to genes for resistance to ZYMV.     

Microsatellite markers of genetic diversity and population structure of Carica papaya

We assessed the genetic diversity of 96 papaya accessions by molecular characterisation using microsatellite markers. Fifteen polymorphic primers were selected. Accessions, which were classified as Common, Formosa and Solo according to fruit types, were evaluated for allele frequency, heterozygosity, polymorphism information content (PIC), inbreeding coefficient (f) and the genetic diversity structure. Fifteen primers amplified 68 alleles with an average of 4.53 per locus. PIC values ranged from 0.19 to 0.69. The observed heterozygosity (H_O) was low for all selected microsatellites. High f estimates (0.58) and excess of homozygotes indicated inbreeding, mainly caused by the tendency to select hermaphrodite plants for succeeding generations. Analysis of molecular variance showed that most of the variation (98%) resides within subpopulation. The genetic analysis based on Bayesian statistics proved to be sensitive enough to detect relationships among the papaya accessions, grouping them into six clusters, irrespective of their classification types.     

Evaluation of cleaved amplified polymorphic sequence markers for <Emphasis Type="Italic">Chamaecyparis obtusa</Emphasis> based on expressed sequence tag information from <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>

We have developed and evaluated sequence-tagged site (STS) primers based on expressed sequence-tag information derived from sugi (Cryptomeria japonica) for use in hinoki (Chamaecyparis obtusa), a species that belongs to a different family (although it appears to be fairly closely related to sugi). Of the 417 C. japonica STS primer pairs we screened, 120 (~30%) were transferable and provided specific PCR amplification products from 16 C. obtusa plus trees. We used haploid megagametophytes to investigate the homology of 80 STS fragments between C. obtusa and C. japonica and to identify orthologous loci. Nearly 90% of the fragments showed high (>70%) degrees of similarity between the species, and 35 STSs indicated homology to entries with the same putative function in a public DNA database. Of the 120 STS fragments amplified, 72 showed restriction fragment length polymorphisms; in addition, the CC2430 primers detected amplicon length polymorphism. We assessed the inheritance pattern of 27 cleaved amplified polymorphic sequence markers, using 20 individuals from the segregation population. All the markers analyzed were consistent with the marker inheritance patterns obtained from the screening panel, and no markers (except CC2716) showed significant (P<0.01) deviation from the expected segregation ratio. In total, 136 polymorphic markers were developed using C. japonica-based STS primers without any sequence modification. In addition, the applicability of STS-based markers developed in one species to other species was found to closely reflect the evolutionary distance between the species, which is roughly concordant with the difference between their rbcL sequences. We plan to use these markers for genetic studies in C. obtusa. Most of the markers should also provide reliable anchor loci for comparative mapping studies of the C. obtusa and C. japonica genomes.     

Development and Characterization of a Genetic Linkage Map of Cryptococcus neoformans var. neoformans Using Amplified Fragment Length Polymorphisms and Other Markers

A segregating population of single basidiospore isolates from a sexual cross was used to generate the first moderately dense genetic linkage map of Cryptococcus neoformans var. neoformans (Serotype D). Polymorphic DNA markers were developed using amplified fragment length polymorphisms, random amplified polymorphic DNA, and gene-encoding sequences. These markers were used to analyze 100 meiotic progeny. All markers were tested for distorted segregation with a goodness of fit test. Of the total of 181 markers, 148 showed balanced (1:1) segregation ratios. Segregation distortion was observed for 33 markers. Based on all the markers, a linkage map was generated that consists of 14 major linkage groups with 127 markers, several small linkage groups, and 2 linkage groups that consist only of highly skewed markers. The genetic distance of the linkage map is 1356.3 cM. The estimated total haploid genome size for C. neoformans var. neoformans was calculated using Hulberts method and yielded a map size of 1917 cM. The number of major linkage groups correlates well with the proposed number of 13 chromosomes for C. neoformans var. neoformans. Several genes, including CAP64, CnLAC, and the mating-type locus, were mapped, and their associations were consistent with published data. To date, 6 linkage groups have been assigned to their corresponding chromosomes. This linkage map should provide a framework for the ongoing genome sequencing project and will be a useful tool for studying the genetics and pathogenicity of this important medical yeast.     

Genetic structure of three populations of rhesus macaques (Macaca mulatta): Implications for genetic management

One of the prime concerns at zoos and at primate breeding facilities is to maintain genetic variability. This can be accomplished by avoiding inbreeding. It is relatively easy to assess genetic variability and the level of inbreeding by using pedigree information and genetic markers. In this study we used genetic markers controlled by 6 independent polymorphic loci (GPI, PGD, CA2, MPI, DIA1, Tf) to ascertain genetic variation in two captive and one wild population of rhesus monkeys. Two other loci ADA and NP were also examined and found to be monomorphic in the three populations. F-statistics and contingency chi-square analyses indicated that there was significant genetic differentiation among the populations. We also found that the mean heterozygosities were very similar in the three populations, in spite of the diverse breeding strategies. These data are important because rhesus monkeys are frequently used for biomedical research; and the genetic markers provide useful information for genetic management of captive colonies of nonhuman primates. © 1992 Wiley-Liss, Inc.     

Correlated pedigree and molecular estimates of inbreeding and their ability to detect inbreeding depression in the Icelandic sheepdog, a recently bottlenecked population of domestic dogs

We examine the relationship of the inbreeding coefficient (f) and molecular estimates of inbreeding (IR, sMLH) in the Icelandic sheepdog. The breed experienced a severe genetic bottleneck and near extinction in the last century. Molecular estimates and the inbreeding coefficient are significantly correlated despite the moderate number of markers and individuals used in this study. This is most likely an effect of the extreme relatedness and relatively high variance in individual inbreeding coefficients in the population. There is a significant relationship between inbreeding (f) and the occurrence of hip dysplasia. However, the molecular measures did not detect hip dysplasia as an inbreeding effect.     

Isolation and characterization of microsatellite markers in the solitary hunting wasp Ancistrocerus adiabatus (Vespidae: Eumeninae) and their cross utility in other species of Ancistrocerus

We have developed seven microsatellite primers for the solitary wasp Ancistrocerus adiabatus. The number of alleles per locus ranged from three to 14. The markers were developed to study population structure, inbreeding and sex determination of A. adiabatus. For each locus, we genotyped 30 female wasps collected from Southwest Michigan. The observed heterozygosity ranges from 0.067 to 0.933. We have also determined that primers for these microsatellite loci amplify in three other species in the genus: Ancistrocerus antilope, Ancistrocerus catskill, and Ancistrocerus campesterus.     

High resolution analysis of mating systems: inbreeding in natural populations of Pinus radiata

Pinus radiata has a history of population bottlenecks and is currently restricted to five relatively small populations, three in mainland California, and two on islands off the coast of Baja California. Using highly polymorphic microsatellite markers and a newly developed statistical approach, we were able to estimate individual inbreeding coefficients and can thus analyse the mating system with high resolution. We find a bimodal distribution of inbreeding coefficients: most individuals result from selfing whereas few (in the mainland populations) to a modest number (in the island populations) are likely selfed. In most other pine species and presumably in the ancestral P. radiata population, occurrence of mature selfed individuals would be impossible because of the high genetic load. We therefore conclude that inbreeding depression has been purged in P. radiata and that the mating system has changed as a consequence.     

Effects of fragmentation phenomena on the genetic structure and gene flow in Centaurea cineraria group (Asteraceae) in the Mediterranean Basin

The complex history of the Mediterranean region illustrates how ancient and recent phenomena are closely associated with species distribution and the creation of phylogeographic divisions within Mediterranean flora. A good model to explore the genetic consequences of fragmentation can be found in Centaurea cineraria and its close relatives. We applied simple sequence repeat molecular markers to a dense population sampling throughout the distribution area of all C. cineraria taxa to study how fragmentation has altered the genetic structure and distribution of C. cineraria. The average gene diversity (H_e) was 0.286, and the average allelic richness (A_r) was 3.65 and ranged from 2.15 (C. gymnocarpa) to 5.25 (C. busambarensis). The F_IS averaged a relatively high 0.223, ranging from ? 0.724 in C. aeolica subsp. aeolica to 0.589 in C. leucadea. Our results indicate that habitat fragmentation over several generations reduced heterozygosity due to random genetic drift in populations of C. cineraria. This heterozygosity erosion becomes more severe when the inbreeding coefficient is positive and the outcrossing rates show a significant increase. The results observed for outcrossing rates and inbreeding coefficient could also indirectly support the possibility of disrupted gene flow or mating pattern changes in fragmented C. cineraria populations.     

Genetic decline and inbreeding depression in an extremely rare tree

Many endangered species have small population sizes, with less than 10 remaining individuals in some extreme situations. Although the consequences of a small population size have received considerable research attention, few studies have examined the fate of extremely rare plants. Ostrya rehderiana is one such species, with only 5 naturally-regenerated surviving individuals and less than 150 artificially-regenerated progeny. Using amplified fragment length polymorphisms (AFLPs), we found that there was a low percentage of polymorphic loci but moderate heterozygosity in the 5 wild individuals. A severe decline in genetic diversity was observed in the progeny, with a decrease of 36.7% in heterozygosity and of 12% in the number of markers that were amplified per individual compared with the parental generation, a result which was caused by genetic drift and inbreeding. The effective population size was estimated to be 1. A significant positive relationship between parental genetic dissimilarity and the number of surviving offspring was observed, which indicated that inbreeding depression might have purged more inbred offspring. Implications for protection and recovery of the genetic variation of extremely rare plants, such as O. rehderiana, are proposed.     

Development of STMS markers from the medicinal plant Madagascar periwinkle [Catharanthus roseus (L.) G. Don.]

We report the isolation and characterization of the first set of sequence‐tagged microsatellites sites (STMS) markers in Catharanthus roseus, a plant with a vast range of medicinal uses. The microsatellite loci were cloned from an enriched library constructed using degenerate primers. Based on the microsatellite motifs, seven STMS primer pairs were designed. They were used to amplify 32 accessions of C. roseus and one accession of Catharanthus trichophyllus. The primers amplified an average of 3.86 alleles per locus. The observed heterozygosity ranged from 0.2903 to 0.9688 with an average of 0.7511. The STMS markers of C. roseus also amplified corresponding loci in a related species (C. trichophyllus) suggesting conservation of the loci across the genus. These markers will prove useful for genetic diversity analysis and linkage map construction in C. roseus.     

Conserved simple sequence repeats for the Limnanthaceae (Brassicales)

The Limnanthaceae (Order Brassicales) is a family of 18 taxa of Limnanthes (meadowfoam) native to California, Oregon, and British Columbia. Cultivated meadowfoam (L. alba Benth.), a recently domesticated plant, has been the focus of research and development as an industrial oilseed for three decades. The goal of the present research was to develop several hundred simple sequence repeat (SSR) markers for genetic mapping, molecular breeding, and genomics research in wild and cultivated meadowfoam taxa. We developed 389 SSR markers for cultivated meadowfoam by isolating and sequencing 1,596 clones from L. alba genomic DNA libraries enriched for AG _n or AC _n repeats, identifying one or more unique SSRs in 696 clone sequences, and designing and testing primers for 624 unique SSRs. The SSR markers were screened for cross- taxa utility and polymorphisms among ten of 17 taxa in the Limnanthaceae; 373 of these markers were polymorphic and 106 amplified loci from every taxon. Cross-taxa amplification percentages ranged from 37.3% in L. douglasii ssp. rosea (145/389) to 85.6% in L. montana (333/389). The SSR markers amplified 4,160 unique bands from 14 genotypes sampled from ten taxa (10.7 unique bands per SSR marker), of which 972 were genotype-specific. Mean and maximum haplotype heterozygosities were 0.71 and 0.90, respectively, among six L. alba genotypes and 0.63 and 0.93, respectively, among 14 genotypes (ten taxa). The SSR markers supply a critical mass of high-throughput DNA markers for biological and agricultural research across the Limnanthaceae and open the way to the development of a genetic linkage map for meadowfoam (x = 5).Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by O. Savolainen     

Identification and characterization of microsatellites for striped bass from repeat-enriched libraries

Striped bass (Morone saxatilis) is economically important in the US due to its value as an aquaculture species and in supporting commercial and recreational fisheries, especially those off the Atlantic coast and in the Gulf of Mexico. Modern strategies for managing fishery populations and aquaculture broodstocks employ the use of molecular genetic markers to identify individuals, assign parentage, and characterize population genetic structure and levels of inbreeding and migration. As part of a collaborative effort to utilize molecular genetic technologies in striped bass breeding programs we generated microsatellite markers for use in population genetic studies, broodstock selection and management strategies, and the construction of a genetic map. We developed 345 new microsatellite markers for striped bass, a subset (n=71) of which was characterized by genotyping samples from two striped bass broodstock populations to evaluate marker polymorphism, percent heterozygosity, Hardy–Weinberg equilibrium (HWE), linkage disequilibrium (LD) and utility for population genetic studies.     

PERMANENT GENETIC RESOURCES: Identification of nuclear microsatellite loci for Ipomopsis aggregata and the distribution of pairwise relatedness in a natural population

Nine microsatellite loci were developed from enriched libraries of scarlet gilia (Ipomopsis aggregata). A screen of 160 individuals from a population identified reduced levels of heterozygosity, low levels of relatedness, and weak spatial genetic patterns. The population inbreeding coefficient was calculated to be 0.19 (SE = 0.04). These patterns are consistent with those expected from low levels of biparental inbreeding in an obligate outcrosser and extensive seed and pollen dispersal. These preliminary data confirm the usefulness of microsatellite markers for breeding system studies of I. aggregata.     

Isolation and characterization of sequence‐tagged microsatellite sites markers in chickpea (Cicer arietinum L.)

In this study we report the isolation of microsatellite sequences and their conversion to sequence‐tagged microsatellite sites (STMS) markers in chickpea (Cicer arietinum L.). Thirteen putative recombinants isolated from a chickpea genomic library were sequenced, and used to design 10 STMS primer pairs. These were utilized to analyse the genetic polymorphism in 15 C. arietinum varieties and two wild varieties, C. echinospermum and C. reticulatum. All the primer pairs amplified polymorphic loci ranging from four to seven alleles per locus. The observed heterozygosity ranged from 0 to 0.6667. Most of the STMS markers also amplified corresponding loci in the wild relatives suggesting conservation of these markers in the genus. Hence, these polymorphic markers will be useful for the evaluation of genetic diversity and molecular mapping in chickpea.