Development of polymorphic microsatellite markers for the zebra finch (Taeniopygia guttata)

To study the population genetics as well as the mating system of captive zebra finch (Taeniopygia guttata) populations, we developed primers for 12 microsatellite loci and screened them in 529 individuals from two successive generations of a single captive population. All markers were polymorphic with five to 14 alleles per locus. We checked all markers for Mendelian inheritance in 307 offspring whose parents were known for sure. Four markers showed evidence for the presence of null alleles. Once allowing for null alleles, we found no mismatches between offspring and parents, suggesting a very low rate of mutation. Average observed and expected heterozygosities across the eight loci showing no evidence for null‐alleles was 0.819 and 0.812, respectively.     

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 polymorphic microsatellite loci from the pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae)

Thirteen polymorphic microsatellite markers were developed from an enriched partial genomic library for the pink bollworm, Pectinophora gossypiella (Saunders), one of the most important cotton pests in the world. The total number of alleles ranged from two to 12 for a sample of 50 individuals and the expected heterozygosity at these loci ranged from 0.042 to 0.830. Although the presence of null alleles in some loci is suspected, these polymorphic markers are likely to provide useful tools for the population genetic studies of this species.     

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.     

Development and characterization of 30 polymorphic microsatellite markers for the Atlantic surfclam, Spisula solidissima (Dillwyn, 1817)

Thirty polymorphic microsatellite markers were developed for the Atlantic surfclam, Spisula solidissima, from an enriched library and characterized in 24 clams from a wild population. The number of alleles ranged from 3 to 16 per locus. The expected and observed heterozygosities ranged from 0.1942 to 0.9238 and 0.0833 to 0.875 respectively. Six loci showed significant (P < 0.05 after Bonferroni correction) deviation from Hardy–Weinberg equilibrium, probably because of the presence of null alleles. Three primer pairs amplified duplicated loci with two involving tandem mini‐satellite repeats. Most of the microsatellite markers developed here should be useful for genetic studies in this species.     

Characterization of five microsatellite loci in the Pine Processionary Moth Thaumetopoea pityocampa (Lepidoptera Notodontidae Thaumetopoeinae)

Five microsatellite markers were developed for the lepidopteran species Thaumetopoea pityocampa using an enrichment protocol. All loci could be amplified with no evidence of null alleles and will be useful for population genetic studies. The number of alleles ranged from three to 12 for a population of 30 individuals. Observed heterozygosities ranged from 0.53 to 0.80. No significant heterozygote deficiency was detected. Four markers might be of interest for Th. wilkinsoni.     

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.     

Polymorphic microsatellite DNA markers for Banksia hookeriana (Proteaceae)

We developed 11 polymorphic microsatellite DNA markers for an Australian native shrub Banksia hookeriana (Proteaceae). The number of alleles per locus in 37 individuals varied from three to 17, observed and expected heterozygosities ranged from 0.297 to 0.838 and from 0.279 to 0.900, respectively. Two loci (BH‐B5 and BH‐B107) showed significant deviation from Hardy–Weinberg equilibrium (P < 0.05), and null alleles may be present at these two loci. All loci showed independent inheritance.     

Nine polymorphic microsatellite markers in Crepis sancta (Asteraceae)

We describe nine new variable di‐ and trinucleotide microsatellites in the plant Crepis sancta, as well as conditions for multiplexing and simultaneous genotyping sets of loci. Their variability was assessed in four populations. Four to 33 alleles were detected per locus; two to 23 alleles per population. Significant deviations from Hardy–Weinberg equilibrium could be attributed either to a mating system involving partial selfing or to null alleles. Genetic differentiation over all loci was significant among the four populations studied. These markers will constitute an efficient tool for investigating dispersal patterns in fragmented landscapes. Cross‐species amplification provided few loci variability in three other Crepis species (C. pulcra, C. foetida and C. rubra).     

Characterization of microsatellite loci in the spruce bark beetle Ips typographus (Coleoptera: Scolytinae)

Ips typographus is an economically important pest of Norway spruce stands. We developed five polymorphic microsatellite markers using a biotin enrichment protocol. The number of alleles ranged from three to 11 per locus and no strong evidence for null alleles was found. Heterozygosity ranged from 0.3 to 0.97. These markers could be useful tools to study the population structure and genetic consequences of I. typographus population outbreaks.     

Discovery of a new fragrance allele and the development of functional markers for the breeding of fragrant rice varieties

The recessive fgr gene on chromosome 8 is associated with rice fragrance. It has been reported that this gene is a non-functional badh2 allele and that the functional Badh2 allele encoding putative betaine aldehyde dehydrogenase (BADH2) could render rice non-fragrant. Here we report the discovery of a new badh2 allele and the development of functional markers for the badh2 locus. A total of 24 fragrant and ten non-fragrant rice varieties were studied and sequenced for their Badh2/badh2 loci. Of the 24 fragrant rice varieties, 12 were found to have the known badh2 allele (badh2-E7), which has an 8-bp deletion and three single nucleotide polymorphisms (SNPs) in exon 7; the others had a novel null badh2 allele (badh2-E2), which has a sequence identical to that of the Badh2 allele in exon 7, but with a 7-bp deletion in exon 2. Both null badh2 alleles are responsible for rice fragrance. Based on sequence divergence amongst the functional Badh2 and two null badh2 alleles, we developed functional markers which can be easily used to distinguish non-fragrant from fragrant rice and to differentiate between two kinds of fragrant rice. These functional markers will find their usefulness in breeding for fragrant rice varieties via marker-assisted selection. Weiwei Shi and Yi Yang contributed equally to this work.     

Microsatellite markers for the whelk Buccinum undatum

Five microsatellite loci are described for the commercially exploited marine gastropod, Buccinum undatum. Levels of polymorphism were variable with three to 19 alleles per locus and expected heterozygosities of 0.26–0.94 in 60 individuals of the population from which the loci were isolated. Homozygote excess at two of the loci might be attributable to null alleles, and these loci should not be used in, for example, parentage analysis. Nevertheless, because null allele frequencies can be estimated and their effects partitioned, all are useful markers for studies of population differentiation.     

Development and characterization of nine polymorphic microsatellite markers in the sand goby Pomatoschistus minutus (Gobiidae)

A microsatellite‐enriched genomic library was constructed for the sand goby, Pomatoschistus minutus (Pallas 1770), and nine polymorphic DNA microsatellite markers of high quality were successfully optimized. Characterization of 96 individuals from the Vaccarès lagoon (France) showed moderate to high levels of polymorphism (two to 54 alleles). All the markers conformed to Hardy–Weinberg equilibrium and showed no evidence of null alleles, large allele dropout, stuttering and linkage disequilibrium between pairs of loci. These markers successfully amplify in three closely related species and can be employed to investigate population genetic structure and to clarify paternity in Pomatoschistus species.     

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.     

DNA-based parentage verification in two Australian goat herds

This study aimed to evaluate a set of DNA markers for their effectiveness in parentage inference, to quantify the level of pedigree errors in Australian Angora and Cashmere goat herds using different pedigree recording methods, and to investigate genotype mismatches between parent and offspring. The 14 microsatellite markers evaluated in this study provided a high level of power (probability of exclusion, PE >99.70%) for parentage testing. The extent of PE depended on polymorphic information content (PIC) and number of alleles for each marker. The minimum number of MS markers essential for accurate determination of parentage was 12, when neither parent is known (PE1) and 10, when one parent is known (PE2). In both populations, the error rates of recorded sire and dam pedigree were significant, averaging around 12%. The error rates of sire and dam pedigree varied considerably between the two populations, reflecting management differences on the two properties. Of 14 MS markers, one locus, SRCRSP07, had null alleles present in the heterozygous state. This null allele was revealed by mismatches of genotypes of parent-offspring pairs. Highly significant deviation from Hardy–Weinberg Equilibrium and significant heterozygote deficiency was also observed at this locus.     

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.     

Polymorphic microsatellite DNA markers in black grouse (Tetrao tetrix)

Ten GATA microsatellite DNA markers were isolated and characterized from black grouse (Tetrao tetrix) using an enrichment cloning procedure. High levels of heterozygosity (mean H_O = 0.74 ± 0.026), and a large number of alleles (range = 5–16) were resolved in 70 individuals, indicating these markers will be useful for examining parentage, inbreeding and population structure in black grouse. No evidence for linkage disequilibrium or the presence of null alleles was found.     

Identification and characterization of microsatellite markers derived from expressed sequence tags of the sea cucumber Stichopus japonicus

Eleven polymorphic microsatellite markers were identified in expressed sequence tags generated from Stichopus japonicus cDNA libraries. The numbers of alleles ranged from three to 10, and the expected and observed heterozygosities ranged from 0.378 to 0.870 and from 0.077 to 0.690, respectively. Significant deviations from Hardy–Weinberg expectations were observed at eight loci due to homozygote excess, suggesting the widespread occurrence of null alleles. The microsatellite markers will be useful for examining genetic population structure, parentage analysis and mapping studies of S. japonicus.     

Characterization of microsatellite DNA markers in the white‐bearded manakin (Manacus manacus)

Eight microsatellite DNA markers were isolated and characterized from white bearded manakin (Manacus manacus) using an enrichment cloning procedure. A large number of alleles (range 9–25), and high levels of observed heterozygosity (mean 0.67) were resolved in 236 individuals. No evidence for linkage disequilibrium or the presence of null alleles was found, indicating that these markers will be useful for examining genetic relatedness, parentage and population structure in manakins.     

Characterization of dinucleotide microsatellite loci in the living fossil tadpole shrimp Triops cancriformis (Crustacea Branchiopoda Notostrace)

Five dinucleotide markers were obtained from a microsatellite genomic library of the tadpole shrimp Triops cancriformis. The number of alleles at each locus was low (maximum three alleles) but all loci were polymorphic. The presence of null alleles could not be disregarded. Heterozygosity levels were indicative of selfing in the studied populations whose real sexuality will be further tested using these markers.