Small sweat bees (Hymenoptera: Halictidae) as potential major pollinators of melon (Cucumis melo) in the Mediterranean

In the current scenario of a general decline of the honeybee worldwide, studies on the potential of alternative bee species in pollinating cultivated plants are important. Although melon, Cucumis melo, is a crop with great commercial importance, there is very little information on its pollinating fauna in Europe, and none from the southern Mediterranean area. In a locality in central Spain, using both pan‐traps and net collections, we found that melon flowers are visited by 31 species of bees spanning four families, though only four were both dominant and constant. These four species belonged to the family Halictidae (sweat bees) and mostly (three species) to the genus Lasioglossum. Five other species could be defined as accessory: honeybee, Apis mellifera, and four other halictids. Individuals of the dominant species were smaller, on average, than those from all the other species. Observations on the frequency of pollen and nectar foraging and on flower visit duration further suggested L. malachurum as the potential key pollinator. Females of this species started to forage on melon early in the flowering season and exhibited two activity peaks in summer, thus covering the whole season. Although in other sites across continents melon seems to be more heavily pollinated by honeybees, this seems to be not the case in the Mediterranean, where sweat bees seem to be the major pollinators of this crop.     

Complex sociogenetic organization and the origin of unrelated workers in a eusocial sweat bee, Lasioglossum malachurum

Sweat bees (Halictidae) exhibit great interspecific and intraspecific diversity in their social organisation, yet there is remarkably little information on the sociogenetic organisation of any species. Lasioglossum malachurum is a eusocial sweat bee with an annual lifecycle that exhibits considerable variation in its social organisation across its wide geographic range from northern to southern Europe. We collected all adults from 31 L. malachurum nests at Eichkogl, Austria, near the latitudinal centre of its distribution, and genotyped 148 workers using 5 highly variable microsatellite loci developed for this species. Nests were often queenless (48% of nests) during the second phase of worker activity, when colonies were provisioning the sexual brood. Pedigree reconstruction and estimates of nestmate genetic relatedness demonstrated that nests often (32% of nests) contained alien workers, probably as a result of worker drifting from their natal to a foreign nest. Queen effective mating frequency was variable (harmonic mean m_e = 1.24), but sometimes high (maximum 2.7). These data demonstrate that nests of L. malachurum do not have a classical eusocial sociogenetic organisation (monogyny, monandry) and thereby pose a challenge to exclusively relatedness based arguments for the evolution of eusociality in the taxon. Received 6 June 2008; revised 1 October 2008; accepted 13 October 2008.     

Development, inheritance and cross-species amplification of microsatellite markers from Acacia mangium

Microsatellite markers were developed in Acacia mangium Willd. to provide highly variable co-dominant markers for linkage mapping and studies of the breeding system. After an enrichment procedure 40% of colonies contained microsatellites in contrast with less than 1% from a non-enriched library. The majority of microsatellite sequences were AC repeats. Co-dominant segregation of alleles in two full-sib crosses of A. mangium was demonstrated at 33 microsatellite loci. The markers were highly variable relative to restriction fragment lengths polymorphisms (RFLPs). In the two pedigrees 53% of microsatellite loci were fully informative compared with 15% of RFLPs. Based on alleles detected among four parental genotypes, the microsatellites consisting of dinucleotide repeats were more polymorphic than those with tri- and tetra-nucleotide repeats. The microsatellite markers were not as transferable across species in the genus Acacia as RFLPs. Two thirds of the primers developed in A. mangium (subgenus Phyllodineae, section Juliflorae) amplified DNA from other species within the same section but failed to amplify in species from the subgenus Acacia. The availability of multiallelic, PCR-based, co-dominant microsatellite loci makes possible efficient studies of gene flow and breeding systems in A. mangium, a species with low allozyme variation. Received: 30 December 1999 / Accepted: 10 May 2000     

Isolation and Characterization of Nine Microsatellite Loci from the Hawaiian Grouper <Emphasis Type="Italic">Epinephelus Quernus</Emphasis> (Serranidae) for Population Genetic Analyses

The availability of variable genetic markers for groupers (Serranidae) has generally been limited to mitochondrial DNA. For studies of population genetic structure, more loci are usually required; particularly useful are those that are nuclear in origin such as microsatellites. Here, we isolated and characterized 9 microsatellite loci from the endemic Hawaiian grouper Epinephelus quernus using a biotin-labeled oligonucleotide-streptavidin–coated magnetic bead approach. Of the 20 repeat-containing fragments isolated, 15 had sufficient flanking region in which to design primers. Among these, 9 produced consistent polymerase chain reaction product, and 6 were highly variable. These 6 loci were all composed of dinucleotide repeats, with the number of alleles ranging from 6 to 18, and heterozygosities from 33.3% to 91.7%. The high levels of variability observed should make these markers useful for population genetic studies of E. quernus, and potentially other epinephelines.     

PCR-RFLP analysis of cpDNA in the genus Abies

 We used PCR-RFLP analysis of the chloroplast DNA of the genus Abies (family Pinaceae), to determine if the method could be employed to detect inter-specific variation in this genus and to study how the variation was distributed in different regions of the genome. Ten different chloroplast DNA regions, consisting of coding and non-coding DNA sequences, were amplified with specific primers in ten different Abies taxa. The amplification products were digested with several restriction enzymes. The results showed that the chloroplast genome is highly variable in most of the investigated taxa and contains multiple variable regions that appear to be distributed throughout the whole genome. Species-diagnostic markers were found for four of the ten investigated species. Unexpectedly, intra-specific variation was also detected in four species. It is likely that further studies, including larger sample sizes and/or more powerful methods for the detection of chloroplast DNA variation, will reveal additional variation for this genus. Received: 2 September 1998 / Accepted: 17 September 1998     

Evaluation of <Emphasis Type="Italic">Hbr</Emphasis> (MITE) markers for assessment of genetic relationships among maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) inbred lines

Recently, a new type of molecular marker has been developed that is based on the presence or absence of the miniature inverted repeat transposable element (MITE) family Heartbreaker (Hbr) in the maize genome. These so-called Hbr markers have been shown to be stable, highly polymorphic, easily mapped, and evenly distributed throughout the maize genome. In this work, we used Hbr-derived markers for genetic characterization of a set of maize inbred lines belonging to Stiff Stalk (SS) and Non-Stiff Stalk (NSS) heterotic groups. In total, 111 markers were evaluated across 62 SS and NSS lines. Seventy six markers (68%) were shared between the two groups, and 25 of the common markers occurred at fairly low frequency (≤0.20). Only two markers (3%) were monomorphic in all samples. Although DNA sequencing indicated that 5.5% of same-sized DNA fragments were non-homologous, this result did not affect the cluster analyses (i.e., relationships obtained from the Hbr data were congruent with those derived from pedigree information). Distance matrices generated from Hbr markers were significantly correlated (p<0.001) with those obtained from pedigree (r=0.782), RFLPs (r=0.747), and SSRs (r=0.719). Overall, these results indicated that Hbr markers could be used in conjunction with other molecular markers for genotyping and relationship studies of related maize inbred lines. Received: 26 February 2001 / Accepted: 20 April 2001     

A highly repeated DNA sequence in Arabidopsis thaliana

Summary Three members of a family of highly repeated DNA sequences from Arabidopsis thaliana have been cloned and characterized. The repeat unit has an average length of 180 bp and is tandemly repeated in arrays longer than 50 kb. This family represents more than one percent of the Arabidopsis genome. Sequence comparisons with tandemly repeated DNA sequences from other Cruciferae species show several regions of homology and a similar length of the repeat unit. Homologies are also found to highly repeated sequences from other plant species. When the sequence CCGG occurs in the repeated DNA, the inner cytosine is generally methylated.     

Study of Resistance of Musa acuminata to Fusarium oxysporum using RAPD markers

Suckers collected from different populations of Musa acuminata ssp. malaccensis were found to be highly resistant to race 4 of Fusarium oxysporum f. sp. cubense (FOC) suggesting that local wild banana populations co-evolved with the pathogen. Seedlings from these wild banana plants segregated for resistance to the pathogen. The infected seedlings were characterized based on external and internal symptoms and the variable response to FOC was mainly due to the genetic factors. Using the technique of random amplified polymorphic DNA (RAPD), 96 major amplification products from 15 primers were identified. Only 10 out of 96 markers were monomorphic and shared among the seed progenies, whereas the remaining 86 were highly polymorphic. Three primers showed banding patterns specific to resistant or susceptible seedlings. These results showed the great potential of the wild Musa acuminata ssp. malaccensis as a source for banana improvement and also for the synthesis of segregating populations for linkage mapping, gene cloning and DNA markers related to FOC resistance.     

Isolation and identification of 21 microsatellite loci in the Copper redhorse (Moxostoma hubbsi; Catostomidae) and their variability in other catostomids

Catostomidae represent an important family of freshwater fishes mainly distributed in North America, but also found in Eurasia. This paper describes the development of microsatellite DNA markers for a highly threatened member of this family, the Copper redhorse (Moxostoma hubbsi), as well as cross‐catostomids amplifications. 168 tetra‐nucleotide loci were screened to develop 21 polymorphic markers, with an average number of 8.5 alleles per locus and observed heterozygosity ranging between 0.52 and 1.00. Successful amplification was obtained for 12 other members of the family at between seven to 19 loci, with between two to 18 loci being polymorphic per species.     

Microsatellite markers for the polygamous termite Nasutitermes corniger (Isoptera: Termitidae)

We developed eight highly variable microsatellite markers for the termite Nasutitermes corniger. Allele number per locus ranged from nine to 34, and expected heterozygosity from 0.45 to 0.94, in samples from seven sites in the former canal zone of Panama. The utility of these markers was assessed for five congeners varying in phylogenetic distance to N. corniger. The markers will be useful for fine‐scale examination of population and colony genetic structure in N. corniger and other closely related species.     

Characterization of dinucleotide microsatellite loci in big brown bats (Eptesicus fuscus), and their use in other North American vespertilionid bats

We describe the development of six microsatellite loci for big brown bats, Eptesicus fuscus. Microsatellite markers were isolated from a small insert genomic library, and tested on a population of 44 animals from the Pend d’Oreille Valley, in southeastern British Columbia, Canada. These six loci were highly variable, with 12–23 alleles per locus, and observed and expected heterozygosities of greater than 79.5%. The six primer sets, and three others that were not variable in E. fuscus, were tested on 11 other species in the families Vespertilionidae and Antrozoidae. All the tested loci amplified highly variable products in at least several other species.     

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.     

The influence of soil temperature on the nesting cycle of the halictid bee Lasioglossum malachurum

The physiology and behavior of ectothermic organisms is strongly influenced by temperature. For ground nesting species like the primitively eusocial halictid bee, Lasioglossum malachurum, soil temperature might influence the life cycle as well as the complexity of the social group since the number of broods that can be fitted into the flight season might increase with increasing temperature. Our study populationof L. malachurum at Wuerzburg exhibits a remarkable variability with respect to the number of broods and the pattern of sexual production. Broods are separated by activity pauses during which the larvae develop. In this study we investigate the influence of soil temperature on the pattern of nesting activity (duration of broods and pauses) and on the number of broods in L. malachurum. We observed a total of 1138 nests in 13 aggregations near Wuerzburg. As expected, soil temperature shortened the duration of the pauses, resulting in an overall shortening of the nesting cycle. This is most probably due to a physiological effect of soil temperature on the development of the larvae. With regard to the nesting strategies, we hypothesized that a shortening of the nesting cycle within the limited flight season should enhance the success of a strategy with more worker broods. In fact, patches with higher soil temperature showed more broods. However, this effect was rather weak, suggesting that other factors might have a stronger impact on the variability in nesting strategy within our study population of L. malachurum. Received 20 December 2005; revised 25 April 2006; accepted 2 May 2006. An erratum to this article is available at .     

Development of 18 polymorphic microsatellite DNA markers of Laminaria japonica (Phaeophyceae)

Eighteen polymorphic microsatellite DNA markers were developed for Laminaria japonica, a brown alga cultured intensively in China. These markers are independent from each other and are moderately variable in L. japonica. The number of alleles and gene diversity detected in 53 gametophyte clones representing the varieties of L. japonica cultured once or currently in China range from two to nine and from 0.355 to 0.768, respectively. These markers will certainly facilitate the management and exploitation of the germplasm resource of L. japonica conserved indoor as gametophyte clones and the determination of the genetic diversity of L. japonica naturally distributed.     

A set of microsatellite primers for the striped possum,Dactylopsila trivirgata (Petauridae: Marsupialia)

The New Guinean endemic dactylopsilines are members of the Petauridae possum family, represented in Australia only by the striped possum Dactylopsila trivirgata. The shy nature and low density of this species have hampered studies of its ecology to date, so we developed nine highly variable polymorphic microsatellite markers to enable molecular genetic analysis of population structure and mating system parameters. This will add substantially to our understanding of the behavioural ecology of this species. The low degree of cross‐amplification of the primers in other petaurid species lends weight to other evidence suggesting that this family underwent a relatively early radiation.     

Characterization and analysis of microsatellite loci in Elymus caninus (Triticeae: Poaceae)

 Microsatellites are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. The potential of microsatellite markers for use in a genetic diversity study in Elymus species was evaluated. Genomic libraries of Elymus caninus were constructed. The libraries were screened with two dinucleotide, (GA)n and (GT)n, and two trinucleotide repeats, (TCT)n and (CAC)n. A total of 19 positive clones were found for the two dinucleotide repeats; no positive clone was found for the trinucleotide repeats. Positive clones were sequenced to confirm the presence of microsatellites and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the microsatellite. All sequenced (GA)n clones have repeats of n>10; over half of the (GT)n microsatellites have n<10 repeats. Primer pairs were designed and evaluated for 8 selected microsatellites. PCR products were amplified from 15 Elymus caninus accessions. The number of alleles found for the eight loci varied from 1 for ECGA89 and ECGT35 to 13 for ECGA22, as determined by non-denaturing polyacrylamide electrophoresis. Six microsatellite loci were found to be polymorphic in E. caninus. The eight primer pairs were tested on three other species; seven were successful in amplifying DNA from Elymus alaskanus and E. mutabilis, and four amplified DNA from E. caucasicus. Based on these results, microsatellites appear to be useful markers in detecting variation in E. caninus. Received: 8 September 1997/Accepted: 6 October 1997     

Structural and functional diversity among bacterial interspersed mosaic elements (BIMEs)

Palindromic units (PU or REP) were defined as 40-nucleotide DNA sequences which are highly repeated in the genome of several members of the Enterobacteriaceae. They were shown to be a constituent of the bacterial interspersed mosaic element (BIME), in which they are associated with other repetitive sequences. We report here that Escherichia coli PU sequences contain three motifs (Y, Z¹ and Z²), leading to the definition of two BIME families. The BIME-1 family, highly conserved over 145 nucleotides, contains two PUs (motifs Y and Z¹). The BIME-2 family contains a variable number of PUs (motifs Y and Z²). We present evidence, using band shift experiments, that each PU motif binds DNA gyrase with a different affinity. This suggests that the two families are functionally distinct.     

Multiple methods for the identification of polymorphic simple sequence repeats (SSRs) in sorghum [Sorghum bicolor (L.) Moench]

Simple sequence repeats (SSRs), also known as microsatellites, are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. Three approaches were followed for the development of PCR primers for the amplification of DNA fragments containing SSRs from sorghum [Sorghum bicolor (L.) Moench]: a search for sorghum SSRs in public DNA databases; the use of SSR-specific primers developed in the Poaceae species maize (Zea mays L.) and seashore paspalum grass (Paspalum vaginatum Swartz); and the screening of sorghum genomic libraries by hybridization with SSR oligonucleotides. A total of 49 sorghum SSR-specific PCR primer pairs (two designed from GenBank SSR-containing sequences and 47 from the sequences of genomic clones) were screened on a panel of 17 sorghum and one maize accession. Ten primer pairs from paspalum and 90 from maize were also screened for polymorphism in sorghum. Length polymorphisms among amplification products were detected with 15 of these primer pairs, yielding diversity values ranging from 0.2 to 0.8 with an average diversity of 0.56. These primer pairs are now available for use as markers in crop improvement and conservation efforts.     

Primers for the amplification of sequence‐characterized loci in Cryphonectria cubensis populations

We describe the development of DNA markers for the fungal pathogen of Eucalyptus, Cryphonectria cubensis. These markers originated from cloned intershort sequence repeat polymerase chain reactions, which enrich for medium to highly repetitive DNA sequences. In total, 10 markers were isolated, eight of which were polymorphic, and these can subsequently be applied to study populations of C. cubensis.     

A set of plastid DNA-specific universal primers for flowering plants

MatK and rbcL are recommended as the official barcode loci for higher plants but there remains a need for additional universal markers. We generated a series of 84 new universal primers targeting 42 plastid loci that all yielded single amplicons when applied to DNA templates from 19 diverse higher plant families. Marker utility ultimately depends on sequence variability, with rapidly evolving loci being useful for barcoding or biogeographic applications and more conserved loci being better suited to deep phylogeny reconstruction. Whereas excessive size variation is undesirable for many applications, modest size variability caused by indels and the sequence variation frequently associated with indels are highly desirable. We therefore performed a quick screen of the markers for size and sequence variation using pooled DNA templates from 96 taxonomically diverse species. All markers produced little or no size variation (consistent with the presence of minor indels). The seven regions exhibiting most size variation in pooled templates (rpl23&rpl2.1, 16S, 23S, 4.5S&5S, petB&D and rpl2, rpoC1 and trnK introns) were then amplified for all species individually, confirming the pooled template results. When the most variable loci (introns of trnK and rpoC1) were sequenced for all 96 species, a high level of sequence variation (nucleotide substitutions and indels) was observed among congeneric species groups for both loci. Both markers therefore have potential as supplementary barcode markers.