Correction method for null alleles in species with variable microsatellite flanking regions, a case study of the black-lipped pearl oyster Pinctada margaritifera

In bivalves, heterozygote deficiencies and departures from Hardy-Weinberg equilibrium (HWE) in microsatellite analysis are common and mainly attributed to inbreeding, genetic patchiness (Walhund effect), or null alleles. We checked for the occurrence of null alleles at 3 microsatellite loci in 3 populations of black-lipped pearl oyster, Pinctada margaritifera, using a step-by-step method to re-amplify homozygotes and null individuals with redesigned primer pair combinations. After amplification with original primer pairs, the 3 populations exhibited null alleles, absence of structure, and significant departure from HWE for all 3 loci due to heterozygote deficiencies. After 3 re-amplification steps, with modified primer sets, all loci were corrected for null alleles. Once corrected, all populations appeared at HWE, demonstrating that null alleles were responsible for the initial disequilibrium of the populations. Furthermore, analysis from corrected genotypes demonstrates significant genetic differentiation for one population from the other 2.     

Genetic control of white flower color in scarlet rosemallow (Hibiscus coccineus Walter)

Scarlet rosemallow (Hibiscus coccineus Walter) is a diploid, perennial, erect, and woody shrub. The species is a desirable inclusion in home landscapes because it is a native plant with attractive flowers and unusual foliage. The objective of these experiments was to determine the number of loci, number of alleles, and gene action controlling flower color (red vs. white) in scarlet rosemallow. Three white-flowered and 1 red-flowered parental lines were used to create S(1) and F(1) populations, which were self-pollinated or backcrossed to generate S(2), F(2), and BC(1) populations. Evaluation of these generations showed that flower color in these populations was controlled by a single diallelic locus with red flower color completely dominant to white. I propose that this locus be named "white flower" with alleles W and w.     

Isolation of nine nuclear microsatellites in the common Mediterranean sea urchin, Paracentrotus lividus (Lamarck)

Nine polymorphic microsatellite loci were isolated and characterized for the edible common sea urchin Paracentrotus lividus. Loci were obtained from two genomic libraries enriched with different di-, tri- and tetranucleotides. Most microsatellites obtained were imperfect dinucleotides. Allelic variation was screened for a total of 56 individuals from two populations from North-Western Mediterranean. Microsatellites showed a number of alleles ranging from 13 to 35. Linkage disequilibrium was not detected between any pair of loci, and all loci showed a significant departure from Hardy-Weinberg equilibrium which is not likely to be the result of null alleles, suggesting that demographic features may be acting upon this commercially interesting 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, characterization, and cross-species utility of microsatellites in yellow cedar (Chamaecyparis nootkatensis).

Chamaecyparis nootkatensis is an ecologically and economically important conifer of the north Pacific coastal forests. To aid in studies of clonal structure and genetic differentiation of this and related species, we isolated and characterized microsatellites from C. nootkatensis. A microsatellite-enriched library yielded 75 repeat-containing sequences for which primer pairs were designed. Only five showed reliable amplification and polymorphism, with an average of 13.7 alleles/locus and a mean expected heterozygosity of 0.592. In progeny tests with four families, few null alleles were directly detected and loci segregated according to Mendelian expectations. However, in one primer pair, high heterozygote deficiency was observed, suggesting the presence of a null allele. The ability of primer pairs to cross amplify was tested on 18 species of the Cupressaceae sensu lato; three primer pairs yielded polymorphic loci in Cupressus and Juniperus species, but not in other Chamaecyparis species. This also supports recent findings of a closer affinity of C. nootkatensis with Cupressus over other Chamaecyparis species.     

A genome-wide SNP genotyping array reveals patterns of global and repeated species-pair divergence in sticklebacks

Genes underlying repeated adaptive evolution in natural populations are still largely unknown. Stickleback fish (Gasterosteus aculeatus) have undergone a recent dramatic evolutionary radiation, generating numerous examples of marine-freshwater species pairs and a small number of benthic-limnetic species pairs found within single lakes [1]. We have developed a new genome-wide SNP genotyping array to study patterns of genetic variation in sticklebacks over a wide geographic range, and to scan the genome for regions that contribute to repeated evolution of marine-freshwater or benthic-limnetic species pairs. Surveying 34 global populations with 1,159 informative markers revealed substantial genetic variation, with predominant patterns reflecting demographic history and geographic structure. After correcting for geographic structure and filtering for neutral markers, we detected large repeated shifts in allele frequency at some loci, identifying both known and novel loci likely contributing to marine-freshwater and benthic-limnetic divergence. Several novel loci fall close to genes implicated in epithelial barrier or immune functions, which have likely changed as sticklebacks adapt to contrasting environments. Specific alleles differentiating sympatric benthic-limnetic species pairs are shared in nearby solitary populations, suggesting an allopatric origin for adaptive variants and selection pressures unrelated to sympatry in the initial formation of these classic vertebrate species pairs.     

Isolation and characterization of polymorphic microsatellites in Parnassius apollo and Euphydryas aurinia (Lepidoptera)

Microsatellite loci were developed from Parnassius apollo and Euphydryas aurinia, two endangered Palaearctic butterfly species. Respectively, six and five polymorphic loci were characterized from an enriched partial genomic library. Genetic diversity range from three to 25 alleles for the first species and from seven to 21 for the second. Although the presence of null alleles is suspected, these polymorphic loci are likely to provide important information on the fine scale genetic structure among populations of these species.     

Characteristics of single- and multi-copy microsatellites from Pinus radiata

 Dinucleotide microsatellites were isolated from Pinus radiata using both a standard genomic library and libraries enriched for microsatellites. Locus-specific primers were designed to amplify 43 unique microsatellites. Thirty two of these loci had interpretable PCR patterns, 11 of which were polymorphic in a screen of 19 P. radiata individuals; all 11 polymorphic loci contained at least 17 repeats in the sequenced plasmid. Six of the eleven primer pairs amplified multiple fragments per individual (3–8), suggesting that these loci were present in multiple copies in the genome. Genotyping a 48-tree P. radiata production population with seven of the most polymorphic microsatellites revealed an average of 17 bands per locus (the multi-copy microsatellites were treated as one locus). When tested on known pedigrees, both single and multi-copy microsatellites exhibited co-dominant inheritance and Mendelian segregation. Two loci had null alleles and one locus had a high frequency of non-parental alleles, suggesting a high mutation rate. Eight of these microsatellites, including five multi-copy loci, were placed on a partially constructed P. radiata genetic map. Four of the five multi-copy microsatellites had two or more sets of alleles that mapped to the same locus, and the fifth mapped to two unlinked loci. All seven tested primer pairs amplified PCR products from other species of hard pine, three amplified products from soft-pine species, and one amplified bands in other conifers. Received: 10 November 1997 / Accepted: 5 January 1998     

Isolation and characterization of ten microsatellite loci for Senegal sole (Solea senegalensis Kaup)

Senegal sole (Solea senegalensis Kaup) is a high‐value marine flatfish exploited in both fisheries and aquaculture. Here we describe the isolation of seven tetranucleotide and three dinucleotide polymorphic microsatellite loci. Characteristics of a captive broodstock and a wild population are described. Three loci displayed a significant departure from Hardy–Weinberg expectations in both populations, suggesting a substantial frequency of null alleles. The remaining seven loci were found to be in equilibrium in the wild population, whereas only four of them were in the cultured population. Cross‐species amplification was successful for three loci in other five flatfishes.     

Silencing of duplicate genes: a null allele polymorphism for lactate dehydrogenase in brown trout (Salmo trutta)

A previously described isozyme polymorphism at one of two skeletal muscle LdhA loci in brown trout is due to a null allele, Ldh1(n), producing no detectable catalytic activity. Homozygotes for this allele have approximately only 56% of the LDH activity in skeletal muscle relative to homozygotes for the active allele. The remaining activity results from enzyme subunits produced by other LDH loci. The Ldh1(n) allele is common and widespread throughout brown trout populations in Sweden and is also found in populations from Ireland. The persistence of duplicate gene expression for the LdhA loci in almost all salmonid species is best explained by natural selection against individuals containing null alleles. However, there is no indication of natural selection against brown trout with the Ldh1(n) allele: We suggest that the selection against individuals containing null alleles that is apparently responsible for the persistence of duplicate LdhA loci in salmonids occurs only under certain environmental conditions.      

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.     

Primers for nine microsatellite loci in the hermaphroditic snail Lymnaea stagnalis

Variation in and amplification conditions for nine polymorphic microsatellite loci identified from Lymnaea stagnalis, a hermaphroditic pulmonate snail, are described. Eight populations from central Finland were studied, which varied in terms of both observed polymorphism and heterozygosity. The number of alleles at each locus is moderate (two to seven), except for one exceptional locus having 16 alleles, and for which null alleles are possible. There is no evidence for genotypic disequilibrium in the populations for all pairs of loci. Heterozygosity levels are indicative of outcrossing in L. stagnalis, whose mating system will be characterized further using these markers.     

Bioinformatic mining of EST-SSR loci in the Pacific oyster, Crassostrea gigas

A set of expressed sequence tag-simple sequence repeat (EST-SSR) markers of the Pacific oyster, Crassostrea gigas, was developed through bioinformatic mining of the GenBank public database. As of June 30, 2007, a total of 5132 EST sequences from GenBank were downloaded and screened for di-, tri- and tetra-nucleotide repeats, with criteria set at a minimum of 5, 4 and 4 repeats for the three categories of SSRs respectively. Seventeen polymorphic microsatellite markers were characterized. Allele numbers ranged from 3 to 10, and the observed and expected heterozygosity values varied from 0.125 to 0.770 and from 0.113 to 0.732 respectively. Eleven loci were at Hardy-Weinberg equilibrium (HWE); the other six loci showed significant departure from HWE (P < 0.01), suggesting possible presence of null alleles. Pairwise check of linkage disequilibrium (LD) indicated that 11 of 136 pairs of loci showed significant LD (P < 0.01), likely due to HWE present in single markers. Cross-species amplification was examined for five other Crassostrea species and reasonable results were obtained, promising usefulness of these markers in oyster genetics.     

Development and Characterization of EST-SSR Markers in the Eastern Oyster <Emphasis Type="Italic">Crassostrea virginica</Emphasis>

Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) in the eastern oyster (Crassostrea virginica). ESTs of the eastern oyster were downloaded from GenBank and screened for SSRs with at least eight units of dinucleotide or five units of tri-, tetra-, penta-, and hexa-nucleotide repeats. The screening of 9101 ESTs identified 127 (1.4%) SSR-containing sequences. Primers were designed for 88 SSR-containing ESTs with good and sufficient flanking sequences. Polymerase chain reaction (PCR) amplification was successful for 71 primer pairs, including 19 (27%) pairs that amplified fragments longer than expected sizes, probably due to introns. Sixty-six pairs that produced fragments shorter than 800 bp were screened for polymorphism in five oysters from three populations via polyacrylamide gels, and 53 of them (80%) were polymorphic. Fifty-three polymorphic SSRs were labeled and genotyped in 30 oysters from three populations via an automated sequencer. Five of the SSRs amplified more than two fragments per oyster, suggesting locus duplication. The remaining 48 SSRs had 2 alleles per individual, including 11 with null alleles. In the 30 oysters analyzed, the SSRs had an average of 9.3 alleles per locus, ranging from 2 to 24. Forty-three loci segregated in a family with 100 progeny, with nine showing significant deviation from Mendelian ratios (three after Bonferroni correction). Seventy percent of the loci were successfully amplified in C. rhizophorae and 34% in C. gigas. This study demonstrates that ESTs are valuable resources for the development of SSR markers in the eastern oyster, and EST-derived SSRs are more transferable across species than genomic SSRs.     

Genetic diversity and population structure of two important medicinal plant species <Emphasis Type="Italic">Schisandra chinensis</Emphasis> and <Emphasis Type="Italic">Schisandra sphenanthera</Emphasis> revealed by nuclear microsatellites

Seven polymorphic and transferable nuclear microsatellites were used to investigate the population structure of genetic diversity of Schisandra chinensis and Schisandra sphenanthera for facilitating their conservation and sustainable utilization. High levels of gene diversity were revealed in these two medicinal species, the majority of genetic diversity was harbored within populations, and population structure was might due to restricted gene flow among populations. Isolation by distance was close to significance in S. chinensis but not in S. sphenanthera. In S. chinensis, null alleles were identified as a cause for excess of homozygotes at loci G24 and WGA60, but inbreeding might also be partly responsible for the positive F _IS values in this species. In contrast, null allele frequencies were high at all the seven loci in S. sphenanthera and resulted in overestimation of fixation index. The strategy for ex situ conservation of these two medicinal species is discussed based on the genetic results.     

Isolation and characterization of polymorphic microsatellite DNA markers in the Omono type of ninespine stickleback,genus Pungitius

We isolated and characterized 25 polymorphic microsatellite loci for the Omono type of ninespine stickleback, genus Pungitius, an endangered species in streams including agricultural canals in Japan. The number of observed alleles per locus ranged from two to 15 within 32 individuals each collected from three populations in the Omono River, and the values of observed and expected heterozygosities ranged from 0.031 to 0.906 and from 0.031 to 0.856, respectively. All loci conformed to the Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed no significant departure from the null hypothesis between any of the loci.     

Comparison of genetic diversity at microsatellite loci in near-extinct and non-endangered species of Mexican goodeine fishes and prediction of cross-amplification within the family

The cross-utility of 12 microsatellite loci (including nine newly developed loci) amongst the viviparous subfamily, the Mexican Goodeinae, was assessed, examining both the probability of amplification and the potential incidence of null alleles from tests of F _IS. Genetic diversity was relatively high in comparison to other freshwater species. Amplification success was not correlated with genetic distance between microsatellite source and target species, but taxa that were more distantly related were less likely to be cross-polymorphic for microsatellite loci. On average, species that were cross-polymorphic were separated by a genetic distance of 15% at the cytochrome oxidase I locus, while those that were monomorphic were separated by 19%. There was no evidence that null alleles become more frequent at greater source-target genetic distance.     

Electrophoretic evidence supporting a theory of allopolyploid origin of the peatmoss Sphagnum jensenii

Based on morphological evidence it has been hypothesised that Sphagnum jensenii is an allopolyploid, with S. annulatum and S. balticum as progenitors. Analysis of nine putative enzyme loci carried out on populations of these three species from central Norway, strongly corroborate this hypothesis. Sphagnum jensenii showed fixed heterozygosity at four of the loci suggesting that it is an allopolyploid. At each of the loci screened, extant populations of S. annulatum and S. balticum shared alleles with S. jensenii . The taxonomic interpretation is that S. jensenii should be recognised as a distinct species different from S. annulatum . Implications of allopolyploidy on ecological tolerances and niche breadth are briefly discussed.     

Reproductive characteristics of the flower breeding Drosophila hibisci Bock (Drosophilidae) along a latitudinal gradient in eastern Australia: relation to flower and habitat features

For many insect species, egg and larval substrate characteristics are significantly correlated with interspecific differences in female reproductive allocation and egg size-number tradeoffs. We tested the hypothesis that a similar pattern occurred within the Australian drosophilid, Drosophila hibisci, that is restricted throughout its life cycle to flowers of species in the genus Hibiscus. These plants occur as small, isolated, normally monospecific stands that should facilitate differentiation of the fly populations in relation to specific oviposition and larval substrates. Data from 38 sites ranging from 20.8̀ to 34.4̀ S latitude in eastern Australia indicated no relationship between female body size, egg size, or ovariole numbers and floral size or mass among four species of Hibiscus. However, the flies did show a latitudinal cline in ovariole number that was independent of floral variation. Females averaged 15–20 ovarioles per female in the south (32–34̀ S latitude) and 10–12 ovarioles in the north (21–22̀ S latitude). The increase in ovariole number with latitude was due to divergence in the ovariole number of the largest females. In contrast, small females in the north and south had the same number of ovarioles. Reproductive allocation of female flies in the northern region was less than females in the southern region. The latitudinal divergence in ovariole number was not associated with habitat differences (density of trees, density of flies and beetles), nor with differences in floral characteristics (flower weight, petal length, yeast species present). Short term weather patterns in daily temperature and rainfall preceding collections pardy explain the variation in ovariole number. These observations in conjunction with preliminary genetic results suggest the cline is associated with genetic differences that interact with environmental determinants such as the temperature during larval development.     

Isolation and characterization of ten polymorphic microsatellite loci in Ixodes arboricola, and cross-amplification in three other Ixodes species

We characterized ten polymorphic microsatellite loci from the tree-hole tick, Ixodes arboricola. Loci were screened in 11–18 individuals from three Belgian populations and five to ten alleles were found at each locus. Seven loci did not show deviations from Hardy–Weinberg equilibrium conditions and there were no indications for null alleles at these loci. The three other loci showed significant heterozygote deficiencies in at least one population, and a high potential for the occurrence of null alleles. We observed no effect of potential host DNA on the scoring of the microsatellites. Cross-amplification of the microsatellites was tested in eight specimens of three congeneric species: I. ricinus, I. hexagonus and I. frontalis. Depending on the species, six or seven of the loci were amplified in ≥4 of the 8 specimens and were polymorphic in each of these species (except for Ixaf 11 in I. frontalis and I. ricinus). These loci thus provide a tool for population genetic analysis of I. arboricola. The suitability of these markers needs to be further investigated in its congeners.