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.     

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.     

Isolation and characterization of microsatellite loci from the endangered highlands scrub hypericum (Hypericum cumulicola)

We report the isolation of 19 primer pairs for amplification of polymorphic microsatellite loci for Hypericum cumulicola. These markers were evaluated in 24 individuals from one population; two to four alleles were detected per locus, and observed heterozygosity ranged from 0 to 0.5. Two loci demonstrated significant heterozygote deficiencies, possibly due to null alleles, and significant linkage disequilibrium was found between six pairs of loci. The remaining microsatellite loci will help determine if genetic differentiation is responsible for life‐history differences between natural and anthropogenically disturbed populations of H. cumulicola.     

Combining multiple analytical approaches for the identification of population structure and genetic delineation of two subspecies of the endemic Arabian burnet moth Reissita simonyi (Zygaenidae; Lepidoptera)

Habitat fragmentation and landscape topology may influence the genetic structure and connectivity between natural populations. Six microsatellite loci were used to infer the population structure of 35 populations (N = 788) of the alpine Arabian burnet moth Reissita simonyi (Lepidoptera, Zygaenidae) in Yemen and Oman. Due to the patchy distribution of larval food plants, R. simonyi is not continuously distributed throughout the studied area and the two recognized subspecies of this endemic species (Reissita s. simonyi/R. s. yemenicola) are apparently discretely distributed. All microsatellites showed prevalence of null alleles and therefore a thorough investigation of the impact of null alleles on different population genetic parameters (F _ST, inbreeding coefficients, and Population Graph topologies) is given. In general, null alleles reduced genetic covariance and independence of allele frequencies resulting in a more connected genetic topology in Population Graphs and an overestimation of pairwise F _ST values and inbreeding coefficients. Despite the presence of null alleles, Population Graphs also showed a much higher genetic connectivity within subspecies (and lower genetic differentiation (via F _ST)) than between; supporting existing taxonomic distinction. Partial Mantel tests showed that both geographical distance and altitude were highly correlated with the observed distribution of genetic structure within R. simonyi. In conclusion, we identified geographical and altitudinal distances in R. simonyi as well as an intervening desert area to be the main factors for spatial genetic structure in this species and show that the taxonomic division into two subspecies is confirmed by genetic analysis.     

Duplicated chromosome segments in maize (Zea mays L.): further evidence from hexokinase isozymes

Summary The genetic control of hexokinase isozymes (ATP: d-hexose-6-phosphotransferase, E.C. 2.7.7.1, HEX) in maize (Zea mays L.) was studied by starch gel electrophoresis. Genetic analysis of a large number of inbred lines and crosses indicates that the major isozymes observed are encoded by two nuclear loci, designated Hex1 and Hex2. Five active allozymes and one null variant are associated with Hex1, while Hex2 has nine active alleles in addition to a null variant. Alleles at both loci govern the presence of single bands, with no intragenic or intergenic heteromers visible, suggesting that maize HEX's are active as monomers. Organelle preparations demonstrate that the products of both loci are cytosolic. All alleles, including the nulls, segregate normally in crosses. Vigorous and fertile plants were synthesized that were homozygous for null alleles at both loci, suggesting that other hexosephosphorylating enzymes exist in maize that are undetected with our assay conditions. Linkage analyses and crosses with B-A translocation stocks place Hex1 on the short arm of chromosome 3, 27 centimorgans from Pgd2 (phosphogluconate dehydrogenase) and Hex2 on the long arm of chromosome 6, approximately 45 centimorgans from Pgd1. It is suggested that the parallel linkages among these two pairs of duplicated genes reflects an evolutionary history involving chromosome segment duplication or polyploidy.Paper No. 10170 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC     

Genetic structure and dispersal in a small South African rodent. Is dispersal female-biased?

Dispersal greatly determines genetic structure of populations, although it is influenced by landscape heterogeneity, quality of the matrix, resource distribution and local population densities and dynamics. To get insights into some of those processes we analysed the genetic structure of the hairy-footed gerbil Gerbillurus paeba (Rodentia, Murinae, Gerbillinae) in the southern Kalahari (South Africa). Samples were taken from 20 populations covering an area of about 2200 km². Genetic data were related to landscape characters and population dynamics. We used newly developed microsatellites and found at all loci some indication for the presence of null alleles. However, null alleles seem to have little influence on the general results of our analyses. Altogether we found even nearby populations of G. paeba to be significantly differentiated, although assignment tests revealed 24% of individuals as immigrants. Genetic structure was independent of landscape heterogeneities at all spatial scales. Autocorrelation analyses (range 50–90 km) revealed significant genetic structure within populations on distances <3 km. We found some indication for female-biased dispersal. Our study suggests that dispersing individuals have little influence on the long-term genetic structure and that drift is the major cause of genetic diversity. The observed genetic pattern likely derives from strong population fluctuations of G. paeba. The landscape structure has little influence on the genetic differentiation between populations.     

Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles sacharovi

The mosquito Anopheles sacharovi, a member of the A. maculipennis complex, is an important malaria vector in the Middle East. Here we describe the isolation of 15 microsatellite polymorphic loci from the A. sacharovi genome, displaying a high among‐individual diversity (0.30–0.92) in a sample from Turkey. Seven loci displayed a significant departure from Hardy–Weinberg proportions, suggesting a substantial frequency of null alleles. The remaining eight loci are good candidates for further genetic studies in this species.     

Isolation and characterization of 11 polymorphic trinucleotide microsatellite markers in the stonefly Arcynopteryx compacta (Plecoptera: Perlodidae)

We describe the isolation of 11 polymorphic trinucleotide microsatellite loci from the stonefly Arcynopteryx compacta. Loci were highly variable with 3 to 14 alleles (mean = 6.45). Observed heterozygosity ranged from 0 to 0.867. Seven loci showed significant deviation from Hardy–Weinberg equilibrium across both populations. There was no evidence for null alleles, and thus, Hardy–Weinberg departures could have resulted from genetic structure between populations or subpopulations. No linkage between loci was found. The 11 loci should prove highly informative for population genetic studies.     

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.     

Genetic diversity within populations of cyanobacteria assessed by analysis of single filaments

We have developed a technique for determining the genetic structure of populations of filamentous cyanobacteria. The sequence diversity at specific gene loci is first characterised in a range of clonal cultures; subsequent analysis involves individual trichomes collected directly from natural populations. This technique has been used to examine the population genetic structure of Nodularia in the Baltic Sea and Planktothrix in Lake Zürich. For Nodularia, studies utilising four polymorphic loci reveal that even though there is a degree of linkage disequilibrium, horizontal transfer of genetic information has been sufficient to generate many of the possible allelic combinations. Analyses reveal both spatial and temporal variation in population genetic structure. Other studies of both Nodularia and Planktothrix have shown a correlation between particular alleles at the gvpC locus and the critical pressure of the gas vesicles that accumulate within the cell. We are now investigating how the natural selection of different gas vesicle phenotypes, imposed by changes in the depth of the upper mixed layer of the water column, affects the relative success of individual cyanobacteria possessing different gvpC alleles. This revised version was published online in August 2006 with corrections to the Cover Date.     

Regional mutagenesis using Dissociation in maize

We describe genetic screens, molecular methods and web resources newly available to utilize Dissociation (Ds) as an insertional mutagen in maize. Over 1700 Ds elements have been distributed throughout the maize genome to serve as donor elements for local or regional mutagenesis. Two genetic screens are described to identify Ds insertions in genes-of-interest (goi). In scheme I, Ds is used to generate insertion alleles when a recessive reference allele is available. A Ds insertion will enable the cloning of the target gene and can be used to create an allelic series. In scheme II, Ds insertions in a goi are identified using a PCR-based screen to identify the rare insertion alleles among a population of testcross progeny. We detail an inverse PCR protocol to rapidly amplify sequences flanking Ds insertion alleles and describe a high-throughput 96-well plate-based DNA extraction method for the recovery of high-quality genomic DNA from seedling tissues. We also describe several web-based tools for browsing, searching and accessing the genetic materials described. The development of these Ds insertion lines promises to greatly accelerate functional genomics studies in maize.     

Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles nili

The mosquito Anopheles nili is widespread across tropical Africa and appears to be the major vector of malaria in some rural forested areas of central Africa. Here we describe the isolation of 11 microsatellite polymorphic loci from the A. nili genome, displaying a high among‐individual diversity (0.58–0.96) in samples from west Africa. Two loci displayed a significant departure from Hardy–Weinberg proportions across all samples, suggesting a substantial frequency of null alleles. The remaining nine loci are good candidates for further genetic studies in this species.     

Assessing the genetic diversity in Argopecten nucleus (Bivalvia: Pectinidae), a functional hermaphrodite species with extremely low population density and self‐fertilization: Effect of null alleles

Argopecten nucleus is a functional hermaphroditic pectinid species that exhibits self‐fertilization, whose natural populations have usually very low densities. In the present study, the genetic diversity of a wild population from Neguanje Bay, Santa Marta (Colombia), was estimated using microsatellite markers, and the effect of the presence of null alleles on this estimation was assessed. A total of 8 microsatellite markers were developed, the first described for this species, and their amplification conditions were standardized. They were used to determine the genotype of 48 wild individuals from Naguanje Bay, and 1,010 individuals derived from the offspring of 38 directed crosses. For each locus, the frequencies of the identified alleles, including null alleles, were estimated using the statistical package Micro‐Checker, and the parental genotypes were confirmed using segregation analysis. Three to 8 alleles per locus with frequencies from 0.001 to 0.632 were detected. The frequencies of null alleles ranged from 0.10 to 0.45, with Ho from 0.0 to 0.79, and He from 0.53 to 0.80. All loci were in H‐W disequilibrium. The null allele frequencies values were high, with lower estimations using segregation analysis than estimated using Micro‐Checker. The present results show high levels of population genetic diversity and indicate that null alleles were not the only cause of deviation from H‐W equilibrium in all loci, suggesting that the wild population under study presents signs of inbreeding and Wahlund effect.     

Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles maculipennis

The Anopheles maculipennis complex includes the most important malaria vectors of the western Palearctic. Anopheles maculipennis s.s., one member of this complex, is a reported vector in the Middle East. Here we describe the isolation of 15 microsatellite polymorphic loci from the An. maculipennis s.s. genome, displaying a high among individual diversity (0.37–0.77) in a sample from France. Three loci displayed a significant departure from Hardy–Weinberg proportions, suggesting a substantial frequency of null alleles. The remaining 12 loci are good candidates for further genetic studies in this species.     

Isolation and characterization of eight polymorphic microsatellite loci for the critically endangered Arenaria nevadensis (Caryophyllaceae)

We report on the isolation and characterization of eight microsatellite markers from enriched libraries for Arenaria nevadensis, one of the most critically endangered plant species in the Iberian Peninsula. These are the first microsatellite loci reported for Arenaria species. The number of alleles ranged from two to eight, and the expected heterozygosity from 0.067 to 0.873. These markers will be useful for characterizing the genetic diversity in A. nevadensis and understanding its population structure, and will provide important genetic data for the conservation and recovery of this species.     

Polymorphic microsatellite loci for the cardinal fish (<Emphasis Type="Italic">Apogon imberbis</Emphasis>)

Eight polymorphic microsatellite loci were isolated and characterized for the Cardinal fish (Apogon imberbis), a coastal-reef fish endemic to the Mediterranean Sea. Characterization of 30 Cardinal fish individuals form the western Mediterranean showed moderate to high allelic diversity ranging from 6 to 19 alleles per locus. Two loci showed significant departures from Hardy-Weinberg equilibrium presumably due to null alleles. No evidence of linkage disequilibrium was found for any locus pairwise comparasions. This microsatellite set could be useful for any basic population genetic studies of this species.     

Isolation and characterization of 10 highly polymorphic di- and trinucleotide microsatellite markers in the mayfly Ameletus inopinatus (Ephemeroptera: Siphlonuridae)

We describe the isolation of ten polymorphic microsatellite loci from the mayfly Ameletus inopinatus. Loci had di‐ or trinucleotide repeat motifs and were highly variable with three to 17 alleles (mean = 7.15). Observed heterozygosity ranged from 0.143 to 0.905. One locus (Ami_202) showed significant deviation from Hardy–Weinberg equilibrium in one population, but no evidence for null alleles. One locus (Ami_73) was significantly linked with three other loci. The remaining nine loci should prove highly informative for population genetic studies.     

Isolation of True Rbp9 Null Alleles by Imprecise P Element Excisions

The P element has been widely used as a mutagen because of its convenience in locating the site of mutagenesis. However, P element-induced mutations often result in varied mutant phenotypes, making it difficult to identify the null phenotype. Previously, three Rbp9 alleles were isolated using P element mutagenesis. Although the coding regions of Rbp9 were disrupted by P elements in all three cases, they showed different degrees of defects. In order to characterize the null phenotype of Rbp9, Rbp9 alleles with chromosomal deletions were created by inducing imprecise excisions of the P elements. All Rbp9 alleles generated from imprecise excisions showed the same mutant phenotype: female flies were sterile and cystocyte differentiation was blocked. This result reveals that the primary function of Rbp9 resides in the regulation of cystocyte differentiation. In addition, this result shows that a P element does not always completely inactivate gene activity, even when it is incorporated into the coding region.     

Population estimators or progeny tests: what is the best method to assess null allele frequencies at SSR loci?

Nuclear SSRs are notorious for having relatively high frequencies of null alleles, i.e. alleles that fail to amplify and are thus recessive and undetected in heterozygotes. In this paper, we compare two kinds of approaches for estimating null allele frequencies at seven nuclear microsatellite markers in three French Fagus sylvatica populations: (1) maximum likelihood methods that compare observed and expected homozygote frequencies in the population under the assumption of Hardy-Weinberg equilibrium and (2) direct null allele frequency estimates from progeny where parent genotypes are known. We show that null allele frequencies are high in F. sylvatica (7.0% on average with the population method, 5.1% with the progeny method), and that estimates are consistent between the two approaches, especially when the number of sampled maternal half-sib progeny arrays is large. With null allele frequencies ranging between 5% and 8% on average across loci, population genetic parameters such as genetic differentiation (F _ST) may be mostly unbiased. However, using markers with such average prevalence of null alleles (up to 15% for some loci) can be seriously misleading in fine scale population studies and parentage analysis.     

Phylogenetic diversity and relationship among Gossypium germplasm using SSRs markers

Gossypium species represent a vast resource of genetic multiplicity for the improvement of cultivated cotton. To determine genetic diversity and relationships within a diverse collection of Gossypium, we employed 120 SSR primers on 20 diploid species representing seven basic genome groups of the genus Gossypium, five AD allotetraploid cotton accessions while T. populnea served as an outgroup species. Out of 120 SSR primers, 49 pairs are polymorphic, which produced a total of 99 distinct alleles with an average of 2.0 alleles per primer pair. A total of 1139 major SSR bands were observed. Genetic similarities among all the diploid species ranged from 0.582 (between G. herbaceum and G. trilobum) up to 0.969 (between G. arboreum and G. herbaceum). Phylogenetic trees based on genetic similarities were consistent with known taxonomic relationships. The results also indicated that G. raimondii is the closest living relative of the ancestral D-genome donor of tetraploid species and the A-genome donor is much similar to the present-day G. herbaceum and G. arboreum. Ancient tetraploid cotton species were formed by hybridizing and chromosome doubling between them, then different tetraploid cotton species appeared by further geographical and genetic isolation and separating differentiation. The results showed that SSRs could be an ideal means for the identification of the genetic diversity and relationship of cotton resources at the genomic level.