Genetic diversity and population structure of 10 Chinese indigenous egg-type duck breeds assessed by microsatellite polymorphism

The genetic structure and diversity of 10 Chinese indigenous egg-type duck breeds were investigated using 29 microsatellite markers. The total number of animals examined were 569, on average 57 animals per breed were selected. The microsatellite marker set analysed provided 177 alleles (mean 6.1 alleles per locus, ranging from 3 to 10). All populations showed high levels of heterozygosity with the lowest estimate of 0.539 for the Jinding ducks, and the highest 0.609 observed for Jingjiang partridge ducks. The global heterozygote deficit across all populations (F _IT) amounted to −0.363. About 10% of the total genetic variability originated from differences among breeds, with all loci contributing significantly. An unrooted consensus tree was constructed using the NeighborNet tree based on the Reynold’s genetic distance. The structure software was used to assess genetic clustering of these egg-type duck breeds. Clustering analysis provided an accurate representation of the current genetic relations among the breeds. An integrated analysis was undertaken to obtain information on the population dynamics in Chinese indigenous egg-type duck breeds, and to better determine the conservation priorities.     

Eighteen novel polymorphic microsatellite loci developed from the Père David’s deer (<Emphasis Type="Italic">Elaphurus davidianus</Emphasis>)

Père David’s deer is a severely bottlenecked species but without showing inbreeding depression, making it essential to develop molecular markers to explore her genetic mechanism of population recovery. In this study, we isolated 18 novel polymorphic microsatellite loci from a dinucleotide-enriched library. This suit of markers presented 2–3 alleles for each locus and their observed and expected heterozygosities were 0.057–0.610 and 0.056–0.598, respectively. These new microsatellite loci had an average of 2.12 alleles and thus contributed to relatively low exclusion probabilities of parentage and paternity testing (0.768 and 0.921). However, when these loci were examined in combination with previous microsatellite markers, overall probabilities of parentage and paternity exclusion went up to 0.905 and 0.990, respectively, showing that these 26 microsatellite loci should be adopted together in future genetic analyses for this highly inbred species.     

Establishment of paternity testing system using microsatellite markers in Chinese Holstein

To estimate the efficiency of microsatellite markers in paternity testing among Chinese Holstein, 30 microsatellite loci were used to differentiate 330 Chinese Holstein genotypes, according to the calculation of the allele frequency, number of alleles, effective number of alleles, genetic heterozygosity, polymorphic information content (PIC), and the exclusion probability in this cattle population. The results demonstrated that the exclusion probability ranged from 0.620 in locus BM1818 to 0.265 in locus INRA005 with the average of 0.472 and 11 microsatellite markers exceeding 0.5. The combined exclusion probability of nine microsatellite markers was over 0.99. The result showed that paternity testing of Chinese Holstein was basically resolved using the nine microsatellite markers selected.     

Isolation and characterization of ten novel microsatellite loci in the red-winged tinamou (<Emphasis Type="Italic">Rhynchotus rufescens</Emphasis>, Tinamiformes,Aves) and cross-amplification in other tinamous

We describe the isolation and characterization of ten microsatellite loci from the red-winged tinamou (Rhynchotus rufescens) and also evaluated the cross-amplification of these loci and other ten loci previously developed for the great tinamou (Tinamus major) in other tinamous. Genetic variability was assessed using 24 individuals. Six loci were polymorphic with moderate to high number of alleles per locus (2–12 alleles) and showed expected heterozygosity (HE) ranging from 0.267 to 0.860. All loci conformed to the Hardy–Weinberg expectation and linkage disequilibrium was not significant for any pair of loci. This battery of polymorphic loci showed high paternity exclusion probability (0.986) and low genetic identity probability (4.95 × 10⁻⁵), proving to be helpful for parentage tests and population analyses in the red-winged tinamou. The cross-amplification was moderate where of the 160 locus/taxon combinations, 46 (28.75%) successfully amplified.     

Microsatellite analysis of genetic diversity in the Chinese alligator (<Emphasis Type="Italic">Alligator sinensis</Emphasis>) Changxing captive population

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. In this study, the extent of genetic variation in the captive alligators of the Changxing Reserve Center was investigated using microsatellite markers derived from American alligators. Out of 22 loci employed, 21 were successfully amplified in the Chinese alligator. Sequence analysis showed loci in American alligators had a bigger average size than that of the Chinese alligators and the longest allele of an individual locus almost always existed in the species with longer stretch of repeat units. Eight of the 22 loci were found to be polymorphic with a total of 26 alleles present among 32 animals scored, yielding an average of 3.25 alleles per polymorphic locus. The expected heterozygosity (H _E) ranged at a moderate level from 0.4385 to 0.7163 in this population. Compared to that in the American alligators, a lower level of microsatellite diversity existed in the Changxing population as revealed by about 46% fewer alleles per locus and smaller H _E at the homologous loci. The average exclusion power and the ability to detect shared genotypes and multiple paternity were evaluated for those markers. Results suggested that when the polymorphic loci were combined, they could be sensitive markers in genetic diversity study and relatedness inference within the Chinese alligator populations. The level of genetic diversity present in the current Changxing population indicated an important resource to complement reintroductions based on the individuals from the other population. In addition, the microsatellite markers and their associated diversity characterized in this population could be utilized to further investigate the genetic status of this species.     

A standardized microsatellite marker panel for parentage and kinship analyses in channel catfish,Ictalurus punctatus

This research was designed to produce a standardized set of microsatellite loci for parentage and kinship analyses in channel catfish, the leading species of US aquaculture. Three panels of five to six markers each were developed that contained a total of two dinucleotide‐, eight trinucleotide‐ and seven tetranucleotide‐microsatellite loci respectively. The loci had a range of nine to 31 alleles per locus in an outbred population. Based on the allele frequencies measured in commercial randomly bred broodstock, the combined probability of non‐exclusion of an unrelated candidate parent pair was 5.36e‐18. The combined probability of non‐exclusion of unrelated identical genotypes was 2.58e‐08. The microsatellite panels were validated by parentage and kinship evaluation in three populations. A total of 697 spawns were collected from matings of outbred broodstock over three spawning seasons, and parents were determined unambiguously for all but three spawns. Genotype analysis also enabled the identification of half‐sibling and full‐sibling families produced by pond spawning. In a second experiment, parentage was unambiguously determined in nine spawns from a population consisting of broodstock derived from only four families. A third experiment demonstrated that all but one of 374 individuals from 10 full‐sibling families could be assigned to a family after coculture in an earthen pond for 1 year. The standardized microsatellite panels enable the development of pedigreed catfish populations and large‐scale performance evaluations in common environments to support the genetic improvement of cultured catfish through selective breeding.     

Single paternity of clutches and sperm storage in the promiscuous green turtle (Chelonia mydas)

Paternity of 22 green turtle ( Chelonia mydas ) clutches from 13 females of the southern Great Barrier Reef breeding population was determined through microsatellite analyses at five loci, including the analysis of successive clutches for nine of the females. A large number of alleles per locus (10–40) provided probabilities of detecting multiple paternity that were quite high, particularly at all loci combined (99.9%). Although green turtles are promiscuous breeders and there was an expectation of finding extensive multiple paternity, only two clutches were multiply sired and, in these, very few eggs had been fertilized by a secondary male. The rarity of multiple paternity may reflect either a low proportion of multiple matings by females in this population, or sperm competition, possibly resulting from a first-male sperm preference. Additionally, the analysis of > 900 offspring provided data on mutations, which included 20 mutation events that were observed in 27 offspring and involved both maternal and paternal lineages. Most mutations ( n = 16) occurred at a single highly variable locus and their presence emphasizes the need to use multiple loci in paternity studies.     

Sixteen novel microsatellite loci developed for the giant panda (Ailuropoda melanoleuca)

Sixteen novel microsatellite DNA loci were developed from the giant panda (Ailuropoda melanoleuca) using a magnetic-bead capture method. A total of 115 alleles were obtained for these markers, ranging from 4 to 12 alleles per locus (average 7.188). These loci exhibited high levels of polymorphic information content and expected heterozygosity, 0.558–0.855 (average 0.729) and 0.628–0.885 (average 0.778), respectively. Therefore, the allelic polymorphism and heterozygosity show that the giant pandas raised in China Research and Conservation Center possess abundant genetic variation. In addition, if the three markers showing null alleles were excluded, the remaining 13 microsatellite loci still presented extremely low non-exclusion probabilities of parentage (0.002), paternity (0.000) and identity (0.000). As a result, this new suit of microsatellite markers would be a very informative tool for the genetic and conservation studies of giant pandas.     

Characterization of paternity relationships in the mole rat <Emphasis Type="Italic">Spalax ehrenbergi</Emphasis> by microsatellite genotyping

Spalax ehrenbergi is a species complex of blind subterranean rodents distributed along the east Mediterranean region. We studied genetic relationships within and between S. ehrenbergi sib families using microsatellite genotyping. The upper-bound level of multiple-paternity rate in litters was estimated using a simulation model of breeding process. Our results show that the upper bound of multiple paternity in the studied population of S. ehrenbergi is <30% (P value 2.9%), with no detected cases of multiple paternity. Our analytical model was specifically designed for a situation in which the sibling identities are known but genetic data about their parents is unavailable. The genetic similarities between groups of individuals were also tested, and it was found that the distance between breeding nests is a factor influencing the genetic similarity between litters found in the nests.     

Eight novel microsatellite markers from the Père David’s deer (<Emphasis Type="Italic">Elaphurus davidianus</Emphasis>)

Père David’s deer underwent known bottlenecks but has recovered to more than 2000 individuals in China, making it interesting to assess its genetic variability from a microsatellite perspective. For this purpose, we developed eight novel microsatellite loci using magnetic-bead enrichment methods. These microsatellite markers revealed a low level of genetic variation in the David’s deer, showing two alleles each locus, 0.31 and 0.38 for average observed and expected heterozyogosities, respectively. Combined with previous cross-species primers, this set of markers would play an important role in future genetic investigation of the David’s deer.     

Paternal genotype reconstruction reveals multiple paternity and sex ratios in a breeding population of leatherback turtles (<Emphasis Type="Italic">Dermochelys</Emphasis><Emphasis Type="Italic">coriacea</Emphasis>)

When animals are difficult to observe while breeding, insights into the mating system may be gained by using molecular techniques. Patterns of extra-pair copulation, multiple paternity and parental genotype analysis may elucidate population characteristics that help improve knowledge of life history while informing management decisions. During the course of a long-term study of leatherback turtles, we assessed the level of multiple paternity in successive clutches for 12 known females nesting at Sandy Point National Wildlife Refuge (St. Croix, U.S. Virgin Islands). We used seven polymorphic microsatellite markers to genotype the females and 1,019 hatchlings representing 38 nests (3–4 clutches from each female). Using deductive genotype reconstruction and GERUD1.0, we identified the 12 mothers and 17 different fathers that were responsible for 38 nests. We found that seven females (58.3%) showed no evidence of multiple paternity in their clutches, while five females (41.7%) had mated with two males each. There was evidence of two fathers (polyandry) in successive clutches for these five females. Multiple fathers didn’t contribute to clutches equally. For clutches laid by an individual female, the primary father was responsible for 53.7 to 85.9% of the hatchlings. We demonstrate the feasibility of using male genotype reconstruction to characterize the male component of this breeding population and to assess operational sex ratios for breeding sea turtles.     

Isolation and characterization of microsatellite loci in Greater Sage‐Grouse (Centrocercus urophasianus)

Primers for five polymorphic microsatellite loci were developed for Greater Sage‐Grouse (Centrocercus urophasianus) using an enrichment/detection protocol. The high level of polymorphism (nine to 33 alleles) suggests that these loci will be applicable for investigating mating systems and paternity analysis as well as population genetics. Cross‐species amplification was successful for each locus in at least two other galliform species.     

Characterization of microsatellite DNA markers in the Emei moustache toads (<Emphasis Type="Italic">Leptobrachium boringii</Emphasis>)

We report ten microsatellite loci in the Emei moustache toads, Leptobrachium boringii. Markers were obtained by screening a genomic library enriched for microsatellite motifs. Twenty-four individuals from one breeding site were examined and ten loci were polymorphic. The number of alleles per locus ranged from 3–9 with an average of 6.3/locus. The expected heterozygosity and observed heterozygosity ranged from 0.3874 to 0.8432, and from 0.4583 to 0.9167, respectively. Cross-species amplification was tested in a closely related species L. leishanensis. These markers will be useful in future studies on characterizing the mating system of the species.     

Development of twelve polymorphic microsatellite loci in polyploid endangered Omphalogramma vincaeflora Franch. (Primulaceae)

A total of twelve polymorphic microsatellite loci were developed from polyploid endangered species, Omphalogramma vincaeflora (Primulaceae). These loci were screened for variability among 45 individuals from three populations in China. The primers amplified loci with allele number ranging from 3 to 9, with an average of 4.25 per locus. Polymorphism information content ranged from 0.23 to 0.86. Nei’s genetic diversity ranged from 0.34 to 0.86. These primers provide an opportunity to use polymorphic DNA markers to study the population genetic structure and its breeding system in this species.     

Paternity testing of wild black rockfish <Emphasis Type="Italic">Sebastes inermis</Emphasis> (brownish type) from the Seto Inland Sea of Japan

We developed four microsatellite DNA loci to test for multiple paternity of black rockfish, Sebastes inermis, from the Seto Inland Sea of Japan. All loci showed a high degree of polymorphism (number of alleles per locus = 10–14, expected heterozygosity = 0.80) and discriminating power (probability of identity index = 3.71 × 10⁻⁶, exclusion probability = 0.999) in unrelated wild specimens (n = 32). Genotypic assignment of five dams (109–220 mm in total length) and 50 embryos from each dam (n = 50) indicated that four dams were mated with a single sire. Only for one dam and three of her embryos we could not exclude multiple paternity.     

Genetic diversity in basmati rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) germplasm as revealed by microsatellite (SSR) markers

Genetic diversity among rice genotypes, including 15 indica basmati advance lines and 5 basmati improved varieties were investigated by 28 SSR markers including one indel marker. The SSRs covered all the 12 chromosomes that distributed across the rice genomes. The mean number of alleles per locus was 3.60, showing average number of polymorphism information content was 0.48. A total of 101 alleles were also identified from the microsatellite marker loci. A number of SSR markers were also identified that could be utilized to differentiate between rice genotypes. Pair wise Nei’s genetic distance between rice genotypes ranged from 0.07 to 0.95. The dendrogram based on cluster analysis by using SSR polymorphism that grouped the 20 genotypes of rice in to five clusters based on their genetic similarity. The result could be useful for the identification and selection of the diverse genotypes for the future cross breeding program and development of new rice varieties.     

Characterization of polymorphic microsatellite loci for the Tawny Pipit Anthus campestris and their utility in other steppe birds

New microsatellite loci for Tawny Pipit Anthus campestris were isolated from a genomic library. We were able to unambiguously score six loci: two were dinucleotide, one trinucleotide, two tetranucleotide and one pentanucleotide that turned out to be sex-linked. Four out of six loci were polymorphic with 7–23 alleles in our population and an observed heterozygosity ranging between 0.286 and 0.936. Cross-utility of these markers was tested in other 17 steppe-bird species of six families. In addition, 16 microsatellite loci developed for other species were tested for cross-species amplification in A. campestris. Eight microsatellite markers were successfully amplified; seven of them were polymorphic with 2–43 alleles and an observed heterozygosity of 0.040–0.863. Overall, 14 functional locus markers have been characterized for A. campestris that could be useful for future studies of paternity, genetic variability and population structure.     

Polymorphic microsatellite loci in the Australian tree frog, <Emphasis Type="Italic">Litoria peronii</Emphasis>

Eleven novel polymorphic microsatellite loci developed from a microsatellite enriched genomic library, are presented for the Australian tree frog Litoria peronii. We screened 29 individuals from a single population and detected high levels of polymorphism for all 11 loci with the number of alleles/locus ranging from 9 to 24. Values of expected and observed heterozygosities ranged from 0.789–0.955 and 0.207–1.00, respectively. These microsatellite markers should prove useful in determining levels of genetic diversity, measuring gene flow and migration, assigning individuals to their most likely population of origin, and in the assignment of paternity.     

Microsatellite markers and multiple paternity in the garter snake thamnophis sirtalis

Data from four microsatellite loci developed for the common garter snake, Thamnophis sirtalis, show that multiple paternity is common in a natural population on Beaver Island, Michigan. Six of eight litters tested, and all litters with five or more neonates, were multiply sired. At least triple paternity was documented in the largest litter examined (n = 13 neonates). Inheritance patterns and genotype frequencies in the wild population indicate the presence of null allele(s) at one of the microsatellite loci. Garter snakes are widely used in quantitative genetics research, and paternity testing is essential in studies that rely on sibling analysis.     

Multiplexed systems of microsatellite markers for genetic analysis of mahogany,Swietenia macrophylla King (Meliaceae), a threatened neotropical timber species

Mahogany (Swietenia macrophylla King [Meliaceae]) is the most valuable hardwood species in the neotropics. Its conservation status has been the subject of increasing concern due to overexploitation and habitat destruction. In this work we report the development and characterization of 10 highly variable microsatellite loci for S. macrophylla. Twenty-nine percent of the 126 sequenced mahogany clones yielded useful microsatellite loci. Three high-throughput genotyping systems were developed based on polymerase chain reaction (PCR) multiplexing of these mahogany loci. We identified a total of 158 alleles in 121 adult individuals of S. macrophylla, with an average of 15.8 alleles (range 11-25) per locus. All loci showed Mendelian inheritance in open-pollinated half-sib families. The mean expected heterozygosity was 0.84 and the mean observed heterozygosity was 0.73. The combined probability of identity-the probability that two individuals selected at random from a population would have identical genotypes--was 7.0 x 10(-15), and combined probability of paternity exclusion was 0.999998 overall loci. These microsatellite loci permit precise estimates of parameters such as gene flow, mating system, and paternity, thus providing important insights into the population genetics and conservation of S. macrophylla.