Microsatellite markers for <Emphasis Type="Italic">Pseudopanax</Emphasis> trees and their cross-species amplification in the Araliaceae

In order to study hybridisation, taxonomic boundaries and phylogeography in the genus Pseudopanax (Araliaceae), we developed seven novel microsatellite loci from enriched genomic libraries of P. lessonii and P. crassifolius. These loci were characterised in 16 individuals from a single population of P. crassifolius, and displayed 2–11 alleles per locus. Observed heterozygosities ranged from 0.063 to 0.688. Most loci were polymorphic in the closely related Pseudopanax species, and several loci amplified widely across the Araliaceae.     

Identification of two loci for resistance to clubroot (<Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis> Woronin) in <Emphasis Type="Italic">Brassica rapa</Emphasis> L.

In an analysis of 114 F₂ individuals from a cross between clubroot-resistant and susceptible lines of Brassica rapa L., 'G004' and 'Hakusai Chukanbohon Nou 7' (A9709), respectively, we identified two loci, Crr1 and Crr2, for clubroot (caused by Plasmodiophora brassicae Woronin) resistance. Each locus segregated independently among the F₂ population, indicating that the loci reside on a different region of chromosomes or on different chromosomes. Genetic analysis showed that each locus had little effect on clubroot resistance by itself, indicating that these two loci are complementary for clubroot resistance. The resistance to clubroot was much stronger when both loci were homozygous for resistant alleles than when they were heterozygous. These results indicate that clubroot resistance in B. rapa is under oligogenic control and at least two loci are necessary for resistance.Communicated by H.C. Becker     

Inheritance of Microsatellite DNA Markers in the Pacific Abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis>

Microsatellite markers have been developed for a variety of abalones, and locus-specific homozygote excesses at population level have been recorded for microsatellite loci. To ascertain whether null alleles exist at microsatellite loci in the Pacific abalone, we studied the mode of inheritance of 7 microsatellite loci in 4 families with a reciprocal cross of 2 females × 2 males. All loci segregated codominantly, but only 3 loci (Hdh1321, Hdh78, and Hdd108C) conformed to Mendelian segregation and can be used for parental analysis and population genetic studies. When null alleles were considered, 2 loci (Hdh1761 and Hdh1457) confirmed Mendelian expectations in all families, while the remaining 2 loci (Hdd114B and Hdd229) showed deviation from Mendelian segregation in at least one family even though null alleles were considered. These results indicated the need to test the inheritance pattern for microsatellite markers in abalones before using them for population genetic or parentage analysis.     

Isolation and characterization of polymorphic microsatellite markers in <Emphasis Type="Italic">Cupressus </Emphasis><Emphasis Type="Italic">chenggiana</Emphasis> S. Y. Hu (Cupressaceae)

Cupressus chenggiana S. Y. Hu (Cupressaceae) is an endemic and endangered conifer species in southwest China. In order to study the population genetics and design the effective conservation methods, we aimed to develop microsatellite primers for this species in the present study. We developed eight new microsatellite loci for this species through biotin capture method. Polymorphism of each locus was further assessed in 18 individuals from three geographically distant populations. The number of alleles per locus ranged from 6 to 11 with an average of 8.13. The observed and expected heterozygosities ranged from 0.219 to 0.296 and from 0.374 to 0.470, with averages of 0.254 and 0.417, respectively. We further found that three of nine microsatellite loci developed previously for another congeneric species showed polymorphic banding patters. We performed primer-crossing tests of these loci in the other two congeneric species which are closely related to C. chenggiana (C. gigantea and C. duclouxiana). These microsatellite markers would be effective for analyzing genetic diversity and population genetic structure of this species and its morphological differentiation with the close relatives.     

Identification and characterization of microsatellite loci in the spiny spider crab <Emphasis Type="Italic">Maja brachydactyla</Emphasis>

The spiny spider crab Maja brachydactyla in an important fishery resource throughout its distribution range (Northeast Atlantic). Here we describe the isolation of nine microsatellite loci for this species. These new markers were tested in 20 crabs from NW Spain. The number of alleles ranged from 3 to 20 and expected heterozigosity from 0.57 to 0.95. All loci followed Hardy–Weinberg expectations except mMb33 and mMb307, which could be affected by null alleles. Some of these loci seem to be better fitted by a multi-step substitution process.     

Larval sex identification in the paper wasp <Emphasis Type="Italic">Polistes dominulus</Emphasis> (Vespidae,Hymenoptera)

Identifying the sex of larvae is important in social Hymenoptera. Until now for Polistes wasps it has been necessary to genotype larvae at microsatellite loci, and assign their sex based on homozygosity at these loci. In our study on the paper wasp Polistes dominulus we have found morphological differences between larval sexes that can be used for larvae from the 3^rd instar on to easily and cheaply identify larval sex: the external gonopore and the shape and size of larval gonads. The robustness of these indicators was supported by genotype data at four microsatellite loci. Using gonopore and gonad features for sex assignment will assign diploid males as males, unlike techniques based on genetic loci or chromosomes. Received 12 July 2006; revised 4 January 2007; accepted 5 February 2007.     

Isolation and characterization of SNP variation at 90 anonymous loci in the banded wren (<Emphasis Type="Italic">Thryothorus pleurostictus)</Emphasis>

Single nucleotide polymorphisms (SNPs) are becoming more commonly used as molecular markers in conservation studies. However, relatively few studies have employed SNPs for species with little or no existing sequence data, partly due to the practical challenge of locating appropriate SNP loci in these species. Here we describe an application of SNP discovery via shotgun cloning that requires no pre-existing sequence data and is readily applied to all taxa. Using this method, we isolated, cloned and screened for SNP variation at 90 anonymous sequence loci (51 kb total) from the banded wren (Thryothorus pleurostictus), a Central American species with minimal pre-existing sequence data and a documented paucity of microsatellite allelic variation. We identified 168 SNPs (a mean of one SNP/305 bp, with SNPs unevenly distributed across loci). Further characterization of variation at 41 of these SNP loci among 256 individuals including 37 parent–offspring families suggests that they provide substantial information for defining the genetic mating system of this species, and that SNPs may be generally useful for this purpose when other markers are problematic.     

Genetic Variation of Microsatellite Loci in the Major Histocompatibility Complex (MHC) Region in the Southeast Asian House Mouse (<Emphasis Type="Italic">Mus musculus castaneus</Emphasis>)

Major histocompatibility complex (MHC) genes are the most polymorphic loci known for vertebrates. Here we employed five microsatellite loci closely linked to the MHC region in an attempt to study the amount of genetic variation in 19 populations of the southeast Asian house mouse (Mus musculus castaneus) in Taiwan. The overall polymorphism at the five loci was high (H_e = 0.713), and the level of polymorphism varied from locus to locus. Furthermore, in order to investigate if selection is operating on MHC genes in natural mouse populations, we compared the extent and pattern of genetic variation for the MHC-linked microsatellite loci (the MHC loci) with those for the microsatellite loci located outside the MHC region (the non-MHC loci). The number of alleles and the logarithm of variance in repeat number were significantly higher for the MHC loci than for the non-MHC loci, presumably reflecting linkage to a locus under balancing selection. Although three statistical tests used do not provide support for selection, their lack of support may be due to low statistical power of the tests, to weakness of selection, or to a profound effect of genetic drift reducing the signature of balancing selection. Our results also suggested that the populations in the central and the southwestern regions of Taiwan might be one part of a metapopulation structure.     

<Emphasis Type="Italic">Tiliqua rugosa</Emphasis> microsatellites: isolation via enrichment and characterisation of loci for multiplex PCR in <Emphasis Type="Italic">T. rugosa</Emphasis> and the endangered <Emphasis Type="Italic">T. adelaidensis</Emphasis>

We used an enrichment technique to isolate 18 novel di and tri microsatellites for the socially monogamous lizard Tiliqua rugosa. These loci were amplified in conjunction with previously described loci in two and three PCR multiplexes for T. rugosa and the endangered T. adelaidensis, respectively. The loci were highly polymorphic in both species, exhibiting between 2 and 32 alleles with observed heterozygosity ranging from 0.43 to 0.96. These markers will be useful for population-level analyses and can contribute to a genetic foundation for conservation strategies for the endangered T. adelaidensis.     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

Development and characterization of novel tetra-, tri- and di-nucleotide microsatellite markers in cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.)

The cacao plant, Theobroma cacao L., produces white seeds (beans) that form the major ingredient of processed chocolate. A great deal of research effort has been expended to the development of new genetically modified cacao plants with improved productivity and resistance and beans of good industrial quality. The availability of suitable genetic markers is an important aspect of the efficient selection and breeding of this perennial species. We describe the development of 123 microsatellite loci of cacao. An optimized protocol was used to construct and screen a microsatellite-enriched genomic library from which we isolated 64 di-nucleotide, 45 tri-nucleotide and 14 tetra-nucleotide microsatellite loci. The primers were tested on samples from five different T. cacao accessions, one accession from T. grandiflorum and one accession from Herranea sp. Among the 123 loci, 54 were polymorphic, 61 were monomorphic and eight did not present an amplification product. These new markers will be useful in future studies by increasing the accuracy of genotypic assessments in diverse cocoa tree populations as well as in other species of the Theobroma genus.     

Sequence,expression divergence,and complementation of homologous <Emphasis Type="Italic">ALCATRAZ</Emphasis> loci in <Emphasis Type="Italic">Brassica napus</Emphasis>

The genomic era provides new perspectives in understanding polyploidy evolution, mostly on the genome-wide scale. In this paper, we show the sequence and expression divergence between the homologous ALCATRAZ (ALC) loci in Brassica napus, responsible for silique dehiscence. We cloned two homologous ALC loci, namely BnaC.ALC.a and BnaA.ALC.a in B. napus. Driven by the 35S promoter, both the loci complemented to the alc mutation of Arabidopsis thaliana, yet only the expression of BnaC.ALC.a was detectable in the siliques of B. napus. Sequence alignment indicated that BnaC.ALC.a and BolC.ALC.a, or BnaA.ALC.a and BraA.ALC.a, possess a high level of similarity. The understanding of the sequence and expression divergence among homologous loci of a gene is of due importance for an effective gene manipulation and TILLING (or ECOTILLING) analysis for the allelic DNA variation at a given locus. S. Hua and I. H. Shamsi contributed equally to this work.     

Isolation and characterization of microsatellite DNA markers for the flagfish <Emphasis Type="Italic">Jordanella </Emphasis><Emphasis Type="Italic">floridae</Emphasis>

The flagfish (Jordanella floridae) is commonly used in studies of wetlands ecology. Here we describe the isolation of ten microsatellite loci, six of which were polymorphic. The observed number of alleles ranged from 4 to 24 and observed heterozygosities ranged from 0.59 to 0.81 for the polymorphic loci. The isolation of these markers will enable estimations of genetic diversity in natural populations.     

Nine microsatellite DNA primers for <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> ssp. <Emphasis Type="Italic">sinensis</Emphasis> (Elaeagnaceae)

Hippophae rhamnoides ssp. sinensis occurs mainly in the arid regions of northwest China. The wood stands of this subspecies play an important role in maintaining the local ecosystems in these regions. In addition, the genetic characteristics are essential to understand the historical range changes of this subspecies and its morphological differentiation with other subspecies. In this study, we developed nine microsatellite loci for this subspecies for the first time. We used the combining biotin capture method to enrich AG/CT/AC/GT/CG/GTG/CCA microsatellites. Twenty-six microsatellites were isolated from the enriching library and nine of them were found to be polymorphic through screening 12 distantly distributed individuals. The number of alleles per locus ranged from three to twelve and expected heterozygosity from 0.2659 to 0.4767, respectively. We further performed cross-priming tests in another subspecies and two congeneric species. These firstly isolated loci will provide a useful tool to investigate the genetic structure of this subspecies and its morphological differentiation from the other subspecies.     

Polymorphic microsatellite loci for species of Australian freshwater cod, <Emphasis Type="Italic">Maccullochella</Emphasis>

The Australian freshwater cod genus, Maccullochella is represented by three species: Murray cod, M. peelii peelii, eastern freshwater cod, M. ikei, and trout cod, M.macquariensis. Seven novel microsatellite loci from M. ikei and six previously published loci from M. peelii peelii were tested on wild populations of Murray, eastern and trout cod. Levels of polymorphism varied between species with 13 loci polymorphic in Murray cod, 9 in trout cod and 7 in eastern cod. Observed heterozygosities ranged from 0.053 to 0.842. This suite of microsatellite loci will facilitate future studies of the genetic status of wild and hatchery bred populations of Maccullochella.     

Validation of the <Emphasis Type="Italic">VRN-H2</Emphasis>/<Emphasis Type="Italic">VRN-H1</Emphasis> epistatic model in barley reveals that intron length variation in <Emphasis Type="Italic">VRN-H1</Emphasis> may account for a continuum of vernalization sensitivity

The epistatic interaction of alleles at the VRN-H1 and VRN-H2 loci determines vernalization sensitivity in barley. To validate the current molecular model for the two-locus epistasis, we crossed homozygous vernalization-insensitive plants harboring a predicted “winter type” allele at either VRN-H1 (Dicktoo) or VRN-H2 (Oregon Wolfe Barley Dominant), or at both VRN-H (Calicuchima-sib) loci and measured the flowering time of unvernalized F₂ progeny under long-day photoperiod. We assessed whether the spring growth habit of Calicuchima-sib is an exception to the two-locus epistatic model or contains novel “spring” alleles at VRN-H1 (HvBM5A) and/or VRN-H2 (ZCCT-H) by determining allele sequence variants at these loci and their effects relative to growth habit. We found that (a) progeny with predicted “winter type” alleles at both VRN-H1 and VRN-H2 alleles exhibited an extremely delayed flowering (i.e. vernalization-sensitive) phenotype in two out of the three F₂ populations, (b) sequence flanking the vernalization critical region of HvBM5A intron 1 likely influences degree of vernalization sensitivity, (c) a winter habit is retained when ZCCT-Ha has been deleted, and (d) the ZCCT-H genes have higher levels of allelic polymorphism than other winterhardiness regulatory genes. Our results validate the model explaining the epistatic interaction of VRN-H2 and VRN-H1 under long-day conditions, demonstrate recovery of vernalization-sensitive progeny from crosses of vernalization-insensitive genotypes, show that intron length variation in VRN-H1 may account for a continuum of vernalization sensitivity, and provide molecular markers that are accurate predictors of “winter vs spring type” alleles at the VRN-H loci.     

Determining colony densities in wild honeybee populations (<Emphasis Type="Italic">Apis mellifera</Emphasis>) with linked microsatellite DNA markers

Estimating the population size of social bee colonies in the wild is often difficult because nests are highly cryptic. Because of the honeybee (Apis mellifera) mating behaviour, which is characterized by multiple mating of queens at drone congregation areas (DCA), it is possible to use genotypes of drones caught at these areas to infer the number of colonies in a given region. However, DCAs are difficult to locate and we assess the effectiveness of an alternative sampling technique to determine colony density based on inferring male genotypes from queen offspring. We compare these methods in the same population of wild honeybees, Apis mellifera scutellata. A set of linked microsatellite loci is used to decrease the frequency of recombination among marker loci and therefore increase the precision of the estimates. Estimates of population size obtained through sampling of queen offspring is significantly larger than that obtained by sampling drones at DCAs. This difference may be due to the more extensive flying range of queens compared with drones on mating flights. We estimate that the population size sampled through queen offspring is about double that sampled through drones.     

Genetic variation and population structure of endemic yellow catfish, <Emphasis Type="Italic">Horabagrus</Emphasis><Emphasis Type="Italic">brachysoma</Emphasis> (Bagridae) among three populations of Western Ghat region using RAPD and microsatellite markers

Random amplified polymorphic DNA (RAPD) and microsatellite markers were applied to evaluate the genetic variation in endemic and endangered yellow catfish, Horabagrus brachysoma sampled from three geographic locations of Western Ghat, South India river systems. In RAPD, of 32 10-mer RAPD primers screened initially, 10 were chosen and used in a comparative analysis of H. brachysoma collected from Meenachil, Chalakkudy and Nethravathi River systems. Of the 124 total RAPD fragments amplified, 49 (39.51%) were found to be shared by individuals of all 3 populations. The remaining 75 fragments were found to be polymorphic (60.48%). In microsatellites, six polymorphic microsatellite loci were identified by using primers developed for Pangasius hypophthalmus, Clarias macrocephalus and Clarias gariepinus. The identified loci were confirmed as microsatellite by sequencing after making a clone. The nucleotide sequences of 6 loci were published in NCBI genbank. The number of alleles across the six loci ranged from 4 to 7 and heterozygosities ranged from 0.07 to 0.93. The mean number of alleles and effective number of alleles per locus were 5.00 and 3.314, respectively. The average heterozygosity across all investigated samples was 0.72, indicating a significant deficiency of heterozygotes in this species. RAPD and microsatellite methods reported a high degree of gene diversity and genetic distances depicted by UPGMA dendrograms among the populations of H. brachysoma.     

Isolation and characterization of 11 polymorphic microsatellite loci from <Emphasis Type="Italic">Fatsia polycarpa</Emphasis> (Araliaceae), an element of evergreen forests in Taiwan

Fatsia polycarpa Hayata (Araliaceae) is an element of evergreen forests at middle elevations in Taiwan, also representing the southern limitation in distribution of the genus. In this study, we described the development of 11 microsatellite loci from F. polycarpa in Taiwan for genetic studies. These new markers were tested in 20 individuals of 5 populations of the woody species. The number of alleles ranged from 9 to 18. The expected (H _E ) and observed (H _O ) heterozygosities were 0.80–0.95 and 0.10–0.75, respectively. Six of the 11 microsatellite loci are significantly deviated from Hardy–Weinberg expectations likely due to the population structure within samples. No linkage disequilibrium were observed from pairwise comparisons of loci. Chi-Chun Huang, Kuo-Hsiang Hung, and Tsai-Wen Hsu contributed equally to this article.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type